Matrix metalloproteinase inhibitors

ABSTRACT

Thus the present invention describes novel compounds comprising 1-10 targeting moieties; a chelator (Ch); and 0-1 linking groups (Ln) between the targeting moiety and chelator; wherein the targeting moiety is a matrix metalloproteinase inhibitor; and wherein the chelator is capable of conjugating to a cytotoxic radioisotope. The present invention also provides novel compositions of the compounds of the invention, kits and their uses in treatment of diseases associated with MMPs.

This application claims the benefit of 60/182,627 filed Feb. 15, 2000.

FIELD OF THE INVENTION

The present invention provides novel pharmaceuticals useful for thediagnosis and treatment of pathologies associated with extracellularmatrix degradation, such as cancer, diabetic retinopathy and maculardegeneration, methods of imaging these pathologies in a patient, andmethods of treating these pathologies in a patient. The invention isalso directed to novel pharmaceutical compositions and combinationtherapy comprising a compound of the invention or a pharmaceuticallyacceptable salt thereof, and at least one agent selected from the groupconsisting of a chemotherapeutic agent and a radiosensitizer agent. Thepharmaceuticals are comprised of a targeting moiety that inhibits amatrix metalloproteinase that is expressed in these pathologies, anoptional linking group, and a therapeutically effective radioisotope ordiagnostically effective imageable moiety. The therapeutically effectiveradioisotope emits a beta particle or alpha particle sufficient to becytotoxic. The imageable moiety is a gamma ray or positron emittingradioisotope, a magnetic resonance imaging contrast agent, an X-raycontrast agent, or an ultrasound contrast agent.

BACKGROUND OF THE INVENTION

Cancer is a major public health concern in the United States and aroundthe world. It is estimated that over 1 million new cases of invasivecancer will be diagnosed in the United States in 1998. The mostprevalent forms of the disease are solid tumors of the lung, breast,prostate, colon and rectum. Cancer is typically diagnosed by acombination of in vitro tests and imaging procedures. The imagingprocedures include X-ray computed tomography, magnetic resonanceimaging, ultrasound imaging and radionuclide scintigraphy. Frequently, acontrast agent is administered to the patient to enhance the imageobtained by X-ray CT, MRI and ultrasound, and the administration of aradiopharmaceutical that localizes in tumors is required forradionuclide scintigraphy.

Treatment of cancer typically involves the use of external beamradiation therapy and chemotherapy, either alone or in combination,depending on the type and extent of the disease. A number ofchemotherapeutic agents are available, but-generally they all sufferfrom a lack of specificity for tumors versus normal tissues, resultingin considerable side-effects. The effectiveness of these treatmentmodalities is also limited, as evidenced by the high mortality rates fora number of cancer types, especially the more prevalent solid tumordiseases. More effective and specific treatment means continue to beneeded.

Despite the variety of imaging procedures available for the diagnosis ofcancer, there remains a need for improved methods. In particular,methods that can better differentiate between cancer and otherpathologic conditions or benign physiologic abnormalities are needed.One means of achieving this desired improvement would be to administerto the patient a metallopharmaceutical that localizes specifically inthe tumor by binding to an enzyme or receptor expressed only in tumorsor expressed to a significantly greater extent in tumors than in othertissue. The location of the metallopharmaceutical could then be detectedexternally either by its imageable emission in the case of certainradiopharmaceuticals or by its effect on the relaxation rate of water inthe immediate vicinity in the case of magnetic resonance imagingcontrast agents.

This tumor specific metallopharmaceutical approach can also be used forthe treatment of cancer when the metallopharmaceutical is comprised of aparticle emitting radioisotope. The radioactive decay of the isotope atthe site of the tumor results in sufficient ionizing radiation to betoxic to the tumor cells. The specificity of this approach for tumorsminimizes the amount of normal tissue that is exposed to the cytotoxicagent and thus may provide more effective treatment with fewerside-effects.

Previous efforts to achieve these desired improvements in cancer imagingand treatment have centered on the use of radionuclide labeledmonoclonal antibodies, antibody fragments and other proteins orpolypeptides that bind to tumor cell surface receptors. The specificityof these radiopharmaceuticals is frequently very high, but they sufferfrom several disadvantages. First, because of their high molecularweight, they are generally cleared from the blood stream very slowly,resulting in a prolonged blood background in the images. Also, due totheir molecular weight they do not extravasate readily at the site ofthe tumor and then only slowly diffuse through the extravascular spaceto the tumor cell surface. This results in a very limited amount of theradiopharmaceutical reaching the receptors and thus very low signalintensity in imaging and insufficient cytotoxic effect for treatment.

Alternative approaches to cancer imaging and therapy have involved theuse of small molecules, such as peptides, that bind to tumor cellsurface receptors. An In-111 labeled somatostatin receptor bindingpeptide, In-111-DTPA-D-Phe¹-octreotide, is in clinical use in manycountries for imaging tumors that express the somatostatin receptor(Baker, et al. Life Sci., 1991, 49, 1583-91 and Krenning, et al., Eur.J. Nucl. Med., 1993, 20, 716-31). Higher doses of thisradiopharmaceutical have been investigated for potential treatment ofthese types of cancer (Krenning, et al., Digestion, 1996, 57, 57-61).Several groups are investigating the use of Tc-99m labeled analogs ofIn-111-DTPA-D-Phe¹-octreotide for imaging and Re-186 labeled analogs fortherapy (Flanagan, et al., U.S. Pat. No. 5,556,939, Lyle, et al., U.S.Pat. No. 5,382,654, and Albert et al., U.S. Pat. No. 5,650,134).

There continues to be a need for more effective treatment options forpatients with solid tumors. This is especially true in cases ofmetastatic cancer in which current standard chemotherapy and externalbeam radiation regimens only result in marginal survival improvements.

Although improvements in cytotoxic chemotherapeutics have been made inrecent years, the toxicity of these compounds to normal tissues hascontinued to severely limit their utility in extending survival inpatients with solid tumors. Recently developed combinations of differenttherapeutic modalities, such as external beam irradiation andchemotherapy (i.e. chemoradiation), has provided some incrementalbenefit to the control of tumor progression and quality of life.However, neither systemic chemotherapeutics nor external beamirradiation have acceptable therapeutic indices, and are often limiteddue to unacceptable toxicity to normal tissues. The concept of combinedtherapy of cancer using anti-angiogenesis drugs in combination withchemotherapeutics is not new. Further, the concept of combining targetedin-vivo radiotherapy using radiolabeled antibodies and antibodyfragments with chemotherapy has been reported (Stein R, Juweid M, ZhangC, et al., Clin. Cancer Res., 5: 3199s-3206s, 1999). However, thecombination of a angiogenesis-targeted therapeutic radiopharmaceuticalwhich is targeted to receptors, which are then upregulated in theneovasculature of tumors, together with chemotherapy has not beendescribed before. Therefore, there is a need for a combination of atherapeutic radiopharmaceutical, which is targeted to localize in theneovasculature of tumors, with chemotherapeutics or a radiosensitizeragent, or a pharmaceutically acceptable salt thereof, to provideadditive or synergistic therapeutic response without unacceptableadditive toxicity in the treatment of solid tumors.

The major advantage of combined chemotherapy and angiogenesis-targetedtherapeutic radiopharmaceuticals, over each therapeutic modality alone,is improved tumor response without substantial increases in toxicityover either treatment alone. The advantage of using neovascular-specificradiopharmaceuticals, versus a tumor-cell targeted antibody, is thatthere is much lower systemic radiation exposure to the subject beingtreated.

Further, if the receptor targets for the radiopharmaceutical compounds,used in this method of treatment, are expressed on the luminal side oftumor vessels, there is no requirement that these compounds traverse thecapillary bed and bind to the tumor itself.

Thus, it is desirable to provide a combination of angiogenesis-targetedtherapeutic radiopharmaceuticals and a chemotherapeutics or aradiosensitizer agent, or a pharmaceutically acceptable salt thereof,which target the luminal side of the neovasculature of tumors, toprovide a surprising, and enhanced degree of tumor suppression relativeto each treatment modality alone without significant additive toxicity.

Matrix metalloproteinases (MMPs) are a family of structurally relatedzinc-containing enzymes that mediate the integrity of extracellularmatrix (Whittaker, M. et al, Chem. Rev., 1999, 99, 2735-2776). They areexcreted by a variety of connective tissue and pro-inflammatory cells,such as, fibroblasts, osteoblasts, macrophages, neutrophils, lymphocytesand endothelial cells. There is now a body of evidence that matrixmetalloproteinases (MMPs) are important in the uncontrolled breakdown ofconnective tissue, including proteoglycan and collagen, leading toresorption of the extracellular matrix. This is a feature of manypathological conditions, such as rheumatoid and osteoarthritis, corneal,epidermal or gastric ulceration; tumor metastasis or invasion;periodontal disease and bone disease. Normally these catabolic enzymesare tightly regulated at the level of their synthesis, as well as, attheir level of extracellular activity through the action of specificinhibitors, such as alpha-2-macroglobulins and TIMP (tissue inhibitor ofmetalloproteinase), which form inactive complexes with the MMPs.Therefore, extracellular matrix degradation and remodeling are regulatedby the relative expression of TIMPs and MMPs. The MMPs are classifiedinto several families based on their domain structure: matrilysin(minimal domain, MMP-7), collagenase (hemopexin domain, MMP-1, MMP-8,MMP-13), gelatinase (fibronectin domain, MMP-2, MMP-9), stromelysin(hemopexin domain, MMP-3, MMP-10, MMP-11), metalloelastase (MMP-12). Inaddition, the transmembrane domain family (MT-MMPs) has been recentlydiscovered and comprises MMP-14 through MMP-17.

It has been established that MMP activity is elevated during tumorprogression. MMPs mediate invasion and metastasis mostly by matrixremodeling, allowing tumor cells to access vessels. MMPs also play arole in primary tumor growth and may be involved in the release ofstroma-bound growth factors and tumor angiogenesis (Summers, J. B., etal, Annual reports in Med. Chem., 1998, 33, 131). MMPs have beendetected in cancerous tissue and the expression of a given MMP is notrestricted to a specific tumor type. Correlation between tumor behaviorand MMP activity in human cancerous tissues has been reported. SomeMMPs, such as the gelatinases are particularly important in tumorprogression.

Therefore, pharmaceuticals targeted to one or more MMP's would be veryuseful for detecting or treating cancerous tissue.

Ahrens, et al. U.S. Pat. No. 5,674,754 discloses methods for thedetection of Matrix Metallo-Proteinase No. 9, using antibodies whichselectively recognize pro-MMP-9 and complexes of pro-MMP-9 with tissueinhibitor of matrix metallo proteinase-1 (TIMP-1), with no substantialbinding to active MMP-9. Venkatesan, et al. U.S. Pat. No. 6,172,057discloses non-peptide inhibitors of matrix metalloproteinases (MMPs) andTNF-.alpha. converting enzyme (TACE) for the treatment of arthritis,tumor metastasis, tissue ulceration, abnormal wound healing, periodontaldisease, bone disease, diabetes (insulinresistance) and HIV infection.

Pathologically, MMPs have been identified as associated with severaldisease states. For example, anomalous MMP-2 levels have been detectedin lung cancer patients, where it was observed that serum MMP-2 levelswere significantly elevated in stage IV disease and in those patientswith distant metastases as compared to normal sera values (Garbisa etal., 1992, Cancer Res., 53: 4548, incorporated herein by reference.)Also, it was observed that plasma levels of MMP-9 were elevated inpatients with colon and breast cancer (Zucker et al., 1993, Cancer Res.53: 140 incorporated herein by reference).

Elevated levels of stromelysin (MMP-3) and interstitial collagenase(MMP-1) have been noted in synovial fluid derived from rheumatoidarthritis patients as compared to post-traumatic knee injury (Walakovitset al., 1992, Arth. Rheum., 35: 35) incorporated herein by reference.Increased levels of mRNA expression for collagenase type I (MMP-1) andcollagenase type IV (MMP-2) have been shown to be increased inulcerative colitis as compared to Crohn's disease and controls (Mattheset al., 1992, Gastroenterology, Abstract 661, incorporated herein byreference). Furthrmore, Anthony et al., 1992, Gastroenterology, Abstract591, demonstrated increased immuno-histochemical expression of thegelatinase antigen in a rabbit model of chronic inflammatory colitis.

It has been shown that the gelatinase MMPs are most intimately involvedwith the growth and spread of tumors. It is known that the level ofexpression of gelatinase is elevated in malignancies, and thatgelatinase can degrade the basement membrane which leads to tumormetastasis. Angiogenesis, required for the growth of solid tumors, hasalso recently been shown to have a gelatinase component to itspathology. Furthermore, there is evidence to suggest that gelatinase isinvolved in plaque rupture associated with atherosclerosis. Otherconditions mediated by MMPs are restenosis, MMP-mediated osteopenias,inflammatory diseases of the central nervous system, skin aging, tumorgrowth, osteoarthritis, rheumatoid arthritis, septic arthritis, cornealulceration, abnormal wound healing, bone disease, proteinuria,aneurysmal aortic disease, degenerative cartilage loss followingtraumatic joint injury, demyelinating diseases of the nervous system,cirrhosis of the liver, glomerular disease of the kidney, prematurerupture of fetal membranes, inflammatory bowel disease, periodontaldisease, age relatedmacular degeneration, diabetic retinopathy,proliferative vitreoretinopathy, retinopathy of prematurity, ocularinflammation, keratoconus, Sjogren's syndrome, myopia, ocular tumors,ocular angiogenesis/neovascularization and corneal graft rejection. Forrecent reviews, see: (1) Recent Advances in Matrix MetalloproteinaseInhibitor Research, R. P. Beckett, A. H. Davidson, A. H. Drummond, P.Huxley and M. Whittaker, Research Focus, Vol. 1, 16-26, (1996), (2)Curr. Opin. Ther. Patents (1994) 4(1): 7-16, (3) Curr. Medicinal Chem.(1995) 2: 743-762, (4) Exp. Opin. Ther. Patents (1995) 5(2): 1087-110,(5) Exp. Opin. Ther. Patents (1995) 5(12): 1287-1196, all of which areincorporated herein by reference.

The P₃′ position is a relatively open area in the succinyl hydroxamates,and a wide range of substitutents, see for example (7), may beintroduced (Sheppard, G. S. et al, Bioorg. Med. Chem. Lett., 1998, 8,3251) at this position. This position also offers the flexibility ofattaching a variety of linkers and chelators for diagnostic purposes.

Therefore, pharmaceuticals targeted to one or more MMPs would be veryuseful for detecting or treating diseases associated with MMPs.

SUMMARY OF THE INVENTION

The pharmaceuticals of the present invention, containing a liganddirected at one or more MMP's (e.g. MMP-1, MMP-2, MMP-3, MMP-9), willlocalize a diagnostic imaging probe or cytotoxic radioisotope to thesite of pathology for the purpose of non-invasive imaging or treatmentof cancerous diseases. The imaging agent may be a MMP inhibitor linkedto radioisotopes which are known to be useful for imaging by gammascintigraphy (Tc-99m, In-111, I-123 and others). Alternatively, the MMPtargeting ligand could be bound to a single or multivalent chelatormoiety. The chelator moiety, in turn, is attached to gadolinium,manganese, or other paramagnetic metal atoms (one or more), which wouldcause a local change in magnetic properties, such as relaxivity orsusceptibility, at the site of tissue damage, which could then be imagedwith magnetic resonance imaging systems. Alternatively, the MMPinhibitor would be bound to a phospholipid or polymer material whichwould be used to encapsulate/stabilize microspheres of gas which wouldbe detectable by ultrasound imaging following localization at the siteof the cancerous tissue.

Imaging agents based on MMP inhibitors would be extremely useful in thedetection, staging and monitoring of tumors. Compounds of the presentinvention, which localize in areas of MMP activity in the tumors andcancerous tissue, will allow detection and localization of suchprocesses which are associated with elevated MMP levels and activity.

Therapeutic radiopharmaceuticals of the present invention comprised of aMMP inhibitor labeled with a radioisotope that emits a beta particle, analpha particle or Auger electrons, localize in tumors selectively anddeliver a cytotoxic dose of radiation to the tumor to treat the disease.

It is one object of the present invention to provide improvedradiopharmaceuticals for the treatment of cancer, comprised of atargeting moiety that inhibits a matrix metalloproteinase that isexpressed in tumors, an optional linking group, and a radioisotopes. Thereceptor binding compounds target the radioisotope to the tumor. Thebeta or alpha-particle or Auger electron emitting radioisotope emits acytotoxic amount of ionizing radiation which results in cell death. Thepenetrating ability of radiation obviates the requirement that thecytotoxic agent diffuse or be transported into the cell to be cytotoxic.

It is another object of the present invention to provide imaging agentsfor pathologies associated with extracellular matrix degradation, suchas cancer, diabetic retinopathy and macular degeneration, comprised ofmatrix metalloproteinase inhibiting compounds conjugated to an imageablemoiety, such as a gamma ray or positron emitting radioisotope, amagnetic resonance imaging contrast agent, an X-ray contrast agent, oran ultrasound contrast agent.

Another aspect of the present invention are diagnostic kits for thepreparation of radiopharmaceuticals useful as imaging agents forpathologies associated with extracellular matrix degradation, such ascancer, diabetic retinopathy and macular degeneration. Diagnostic kitsof the present invention comprise one or more vials containing thesterile, non-pyrogenic, formulation comprised of a predetermined amountof a reagent of the present invention, and optionally other componentssuch as one or two ancillary ligands, reducing agents, transfer ligands,buffers, lyophilization aids, stabilization aids, solubilization aidsand bacteriostats. The inclusion of one or more optional components inthe formulation will frequently improve the ease of synthesis of theradiopharmaceutical by the practicing end user, the ease ofmanufacturing the kit, the shelf-life of the kit, or the stability andshelf-life of the radiopharmaceutical. The inclusion of one or twoancillary ligands is required for diagnostic kits comprising reagentcomprising a hydrazine or hydrazone bonding moiety. The one or morevials that contain all or part of the formulation can independently bein the form of a sterile solution or a lyophilized solid.

Another aspect of the present invention contemplates a method of imagingpathologies associated with extracellular matrix degradation, such ascancer, diabetic retinopathy and macular degeneration in a patientinvolving: (1) synthesizing a diagnostic radiopharmaceutical of thepresent invention, using a reagent of the present invention, capable oflocalizing in sites of extracellular matrix degradation; (2)administering said radiopharmaceutical to a patient by injection orinfusion; (3) imaging the patient using planar or SPECT gammascintigraphy, or positron emission tomography.

Another aspect of the present invention contemplates a method of imagingpathologies associated with extracellular matrix degradation, such ascancer, diabetic retinopathy and macular degeneration in a patientinvolving: (1) administering a paramagnetic metallopharmaceutical of thepresent invention capable of localizing in sites of extracellular matrixdegradation to a patient by injection or infusion; and (2) imaging thepatient using magnetic resonance imaging.

Another aspect of the present invention contemplates a method of imagingpathologies associated with extracellular matrix degradation, such ascancer, diabetic retinopathy and macular degeneration in a patientinvolving: (1) administering a X-ray contrast agent of the presentinvention capable of localizing in sites of extracellular matrixdegradation to a patient by injection or infusion; and (2) imaging thepatient using X-ray computed tomography.

Another aspect of the present invention contemplates a method of imagingpathologies associated with extracellular matrix degradation, such ascancer, diabetic retinopathy and macular degeneration in a patientinvolving: (1) administering a ultrasound contrast agent of the presentinvention capable of localizing in sites of extracellular matrixdegradation to a patient by injection or infusion; and (2) imaging thepatient using sonography.

Another aspect of the present invention contemplates a method oftreating pathologies associated with extracellular matrix degradation,such as cancer, diabetic retinopathy and macular degeneration in apatient involving: (1) administering a therapeutic radiopharmaceuticalof the present invention capable of localizing in sites of extracellularmatrix degradation to a patient by injection or infusion.

DETAILED DESCRIPTION OF THE INVENTION

Thus the present invention includes the following embodiments:

(1) A compound comprising:

-   -   i) 1-10 targeting moieties;    -   ii) a chelator (Ch); and    -   iii) 0-1 linking groups (Ln) between the targeting moiety and        chelator;        wherein the targeting moiety is a matrix metalloproteinase        inhibitor; and        wherein the chelator is capable of conjugating to a cytotoxic        radioisotope.

(2) A compound according to embodiment 1, wherein the targeting moietyis a matrix metalloproteinase inhibitor having an inhibitory constantK_(i) of <1000 nM.

(3) A compound according to embodiment 1, wherein the targeting moietyis a matrix metalloproteinase inhibitor having an inhibitory constantK_(i) of <100 nM.

(4) A compound according to any one of embodiments 1-3, comprising 1-5targeting moieties.

(5) A compound according to embodiment 1, comprising one targetingmoiety.

(6) A compound according to any one of embodiments 1-5, wherein thetargeting moiety is a matrix metalloproteinase inhibitor of the formulae(Ia) or (Ib):

wherein,

-   R is independently OH or —CH₂SH;-   R¹ is independently selected at each occurrence from the group: H,    OH, C₁₋₃ alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl, and heterocycle-S—CH₂—;-   R² is independently C₁₋₂₀ alkyl;-   X is independently C═O or SO₂, provided when X is C═O, R³ is    and when X is SO₂, R³ is independently selected from the group: aryl    substituted with 0-2 R⁶, and heterocycle substituted with 0-2 R⁶;-   R⁴ is independently selected at each occurrence from the group: C₁₋₆    alkyl, phenyl, and benzyl;-   R⁵ is independently at each occurrence from the group: NH(C₁₋₆    alkyl), NH-phenyl, and NH-heterocycle; wherein said alkyl, phenyl    and heterocycle groups are optionally substituted with a bond to the    linking group or a bond to the chelator;-   R⁶ is independently aryloxy substituted with 0-3 R⁷;-   R⁷ is independently halogen or methoxy;    or alternatively,-   R¹ and R⁴ may be taken together to form a bridging group of the    formula —(CH₂)₃—O-phenyl-CH₂—, optionally substituted with a bond to    the linking group or a bond to the chelator;    or alternatively,-   R¹ and R² may be taken together to form a bridging group of the    formula —(CH₂)₃—NH—, optionally substituted with a bond to the    linking group or a bond to the chelator; or-   R¹ and R² taken together with the nitrogen and carbon atom through    which they are attached form a C₅₋₇ atom saturated ring system    substituted with one or more substituents selected from the group    consisting of: a bond to Ln, a bond to C_(h), and —C(═O)—NR²⁹R³⁰;-   R⁸ is independently at each occurrence OH or phenyl, optionally    substituted with a bond to the linking group or a bond to the    chelator, provided that when R⁸ is phenyl, R¹⁰ is    —C(═O)—CR¹²—NH—CH(CH₃)—COOH;-   R⁹ and R⁹′ are independently H, C₁₋₆ alkyl optionally substituted    with a bond to the linking group or a bond to the chelator, or are    taken together with the carbon atom to which R⁹ and R⁹′ are attached    to form a 5-7 atom saturated, partially unsaturated or aromatic ring    system containing 0-3 heteroatoms selected from O, N, SO₂ and S,    said ring system substituted with R⁶ and optionally substituted with    a bond to the linking group or a bond to the chelator;

R¹⁰ and R¹¹ are independently H, or C₁₋₆ alkyl optionally substitutedwith a bond to the linking group or a bond to the chelator, or are takentogether with the nitrogen atom to which they are attached to form a 5-7atom saturated, partially unsaturated or aromatic ring system containing0-3 heteroatoms selected from O, N, SO₂ and S, said ring systemoptionally substituted with 0-3 R²⁷, a bond to the linking group or abond to the chelator;

or alternatively,

R⁹ and R¹⁰ are taken together with the carbon atom to which they areattached to form a 5-7 atom saturated, partially unsaturated or aromaticring system containing 0-3 heteroatoms selected from O, N, SO₂ and S,said ring system optionally substituted with a bond to the linking groupor a bond to the chelator; and

-   R¹² is independently C₁₋₂₀ alkyl;-   R²⁷ is ═O, C1-4 alkyl, or phenyl substituted with R²⁸;-   R²⁸ is a phenoxy group substituted with 0-2 OCH₃ groups;-   R²⁹ and R³⁰ taken together with the nitrogen atom through which they    are attached form a C5-7 atom saturated ring system substituted with    R³¹; and-   R³¹ is a benzyloxy group substituted with C1-4 alkyl.

(7) A compound according to any one of embodiments 1-6 wherein thetargeting moiety is a matrix metalloproteinase inhibitor of the formulae(Ia) or (Ib):

wherein,

-   R is OH;-   R¹ is independently selected at each occurrence from the group: H,    OH, C₁₋₃ alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl, and heterocycle-S—CH₂—;-   R² is independently C₁₋₆ alkyl;-   X is C═O;-   R⁴ is independently selected at each occurrence from the group: C₁₋₆    alkyl, phenyl, and benzyl;-   R⁵ is independently at each occurrence from the group: NH(C₁₋₆    alkyl), NH-phenyl, and NH-heterocycle; wherein said alkyl, phenyl    and heterocycle groups are optionally substituted with a bond to the    linking group or a bond to the chelator;-   R⁶ is independently aryloxy substituted with 0-3 R⁷;-   R⁷ is independently halogen or methoxy;    or alternatively,-   R¹ and R⁴ may be taken together to form a bridging group of the    formula —(CH₂)₃—O-phenyl-CH₂—, optionally substituted with a bond to    the linking group or a bond to the chelator;    or alternatively,-   R¹ and R² may be taken together to form a bridging group of the    formula —(CH₂)₃—NH—, optionally substituted with a bond to the    linking group or a bond to the chelator; or-   R¹ and R² taken together with the nitrogen and carbon atom through    which they are attached form a C₅₋₇ atom saturated ring system    substituted with one or more substituents selected from the group    consisting of: a bond to Ln, a bond to C_(h), and —C(═O)—NR²⁹R³⁰;-   R⁸ is OH;-   R⁹ and R⁹′ are independently H, C₁₋₆ alkyl optionally substituted    with a bond to the linking group or a bond to the chelator, or are    taken together with the carbon atom to which R⁹ and R⁹′ are attached    to form a 5-7 atom saturated, partially unsaturated or aromatic ring    system containing 0-1 heteroatoms selected from O, N, said ring    system optionally substituted with a bond to the linking group or a    bond to the chelator,-   R¹⁰ and R₁₁ are independently H, or C₁₋₆ alkyl optionally    substituted with a bond to the linking group or a bond to the    chelator, or are taken together with the nitrogen atom to which they    are attached to form a 5-7 atom saturated, partially unsaturated or    aromatic ring system containing 0-1 heteroatoms selected from O, N,    said ring system optionally substituted with 0-3 R²⁷, a bond to the    linking group or a bond to the chelator;    or alternatively,-   R⁹ and R¹⁰ are taken together with the carbon atom to which they are    attached to form a 5-7 atom saturated, partially unsaturated or    aromatic ring system containing 0-1 heteroatoms selected from O, N,    said ring system optionally substituted with a bond to the linking    group or a bond to the chelator; and-   R¹² is independently C₁₋₆ alkyl;-   R²⁷ is ═O, C1-4 alkyl, or phenyl substituted with R²⁸;-   R²⁸ is a phenoxy group substituted with 0-2 OCH₃ groups;-   R²⁹ and R³⁰ taken together with the nitrogen atom through which they    are attached form a C5-7 atom saturated ring system substituted with    R³¹; and-   R³¹ is a benzyloxy group substituted with C1-4 alkyl.

(8) A compound according to any one of embodiments 1-7 wherein:

-   R is —OH;-   R² is C₁₋₆ alkyl;-   X is C═O;-   R³ is

R¹ and R⁴ are taken together to form a bridging group of formula—(CH₂)₃—O-phenyl-CH₂—;

-   R⁵ is NH(C1-6alkyl), substituted with a bond to the linking group or    a bond to the chelator.-   A compound according to any one of embodiments 1-8, wherein:-   R is —OH;-   R⁹ is C₁ alkyl substituted with a bond to Ln;-   R¹⁰ and R¹¹ taken together with the nitrogen atom to which they are    attached form a 5 atom saturated ring system, said right system is    substituted with 0-3 R²⁷;-   R²⁷ is ═O, C1-4 alkyl, or phenyl substituted with R²⁸; and-   R²⁸ is a phenoxy group substituted with 0-2 OCH₃ groups.

(9) A compound according to any one of embodiments 1-8, wherein:

-   R is —OH; R¹ and R² taken together with the nitrogen and carbon atom    through which they are attached form a C₅₋₇ atom saturated ring    system substituted with one or more substituents selected from the    group consisting of: a bond to Ln, a bond to C_(h), and    —C(═O)—NR²⁹R³⁰; R²⁹ and R³⁰ taken together with the nitrogen atom    through which they are attached form a C5-7 atom saturated ring    system substituted with R³¹; and R³¹ is a benzyloxy group    substituted with C1-4 alkyl.

(10) A compound according to any one of embodiments 1-9, wherein thelinking group is of the formula:((W¹)_(h)—(CR¹³R¹⁴)_(g))_(x)—(Z)_(k)—((CR^(13a)R^(14a))_(g′)—(W²)_(h′))_(x′;)

-   W¹ and W² are independently selected at each occurrence from the    group: O, S, NH, NHC(═O), C(═O)NH, NR¹⁵C(═O), C(═O)NR¹⁵, C(═O),    C(═O)O, OC(═O), NHC(═S)NH, NHC(═O)NH, SO₂, SO₂NH, —(OCH₂CH₂)₇₆₋₈₄,    (OCH₂CH₂)_(s), (CH₂CH₂O)_(s′), (OCH₂CH₂CH₂)_(s″), (CH₂CH₂CH₂O)_(t),    and (aa)_(t′;)-   aa is independently at each occurrence an amino acid;-   Z is selected from the group: aryl substituted with 0-3 R¹⁶, C₃₋₁₀    cycloalkyl substituted with 0-3 R¹⁶, and a 5-10 membered    heterocyclic ring system containing 1-4 heteroatoms independently    selected from N, S, and O and substituted with 0-3 R¹⁶;-   R^(13a), R¹⁴, R^(14a), and R¹⁵ are independently selected at each    occurrence from the group: H, ═O, COOH, SO₃H, PO₃H, C₁-C₅ alkyl    substituted with 0-3 R¹⁶, aryl substituted with 0-3 R¹⁶, benzyl    substituted with 0-3 R¹⁶, and C₁-C₅ alkoxy substituted with 0-3 R¹⁶,    NHC(═O)R¹⁷, C(═O)NHR¹⁷, NHC(═O)NHR¹⁷, NHR¹⁷, R¹⁷, and a bond to the    chelator;-   R¹⁶ is independently selected at each occurrence from the group:    -   a bond to the chelator, COOR¹⁷, C(═O)NHR¹⁷, NHC(═O)R¹⁷, OH,        NHR¹⁷, SO₃H, PO₃H, —OPO₃H₂, —OSO₃H, aryl substituted with 0-3        R¹⁷, C₁₋₅ alkyl substituted with 0-1 R¹⁸, C₁₋₅ alkoxy        substituted with 0-1 R¹⁸, and a 5-10 membered heterocyclic ring        system containing 1-4 heteroatoms independently selected from N,        S, and O and substituted with 0-3 R¹⁷;-   R¹⁷ is independently selected at each occurrence from the group: H,    alkyl substituted with 0-1 R¹⁸, aryl substituted with 0-1 R¹⁸, a    5-10 membered heterocyclic ring system containing 1-4 heteroatoms    independently selected from N, S, and O and substituted with 0-1    R¹⁸, C₃₋₁₀ cycloalkyl substituted with 0-1 R¹⁸, polyalkylene glycol    substituted with 0-1 R¹⁸, carbohydrate substituted with 0-1 R¹⁸,    cyclodextrin substituted with 0-1 R¹⁸, amino acid substituted with    0-1 R¹⁸, polycarboxyalkyl substituted with 0-1 R¹⁸, polyazaalkyl    substituted with 0-1 R¹⁸, peptide substituted with 0-1 R¹⁸, wherein    the peptide is comprised of 2-10 amino acids,    3,6-O-disulfo-B-D-galactopyranosyl, bis(phosphonomethyl)glycine, and    a bond to the chelator;-   R¹⁸ is a bond to the chelator;-   k is selected from 0, 1, and 2;-   h is selected from 0, 1, and 2;-   h′ is selected from 0, 1, and 2;-   g is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   g′ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   s is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   s′ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   s″ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   t is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   t′ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   x is selected from 0, 1, 2, 3, 4, and 5; and-   x′ is selected from 0, 1, 2, 3, 4, and 5.

11. A compound according to any one of embodiments 6-10 wherein W¹ andW² are independently selected at each occurrence from the group: O, NH,NHC(═O), C(═O)NH, NR¹⁵C(═O), C(═O)NR¹⁵, C(═O), C(═O)O, OC(═O),NHC(═S)NH, NHC(═O)NH, SO₂, —(CH₂CH₂O)₇₆₋₈₄-, (OCH₂CH₂)_(s),(CH₂CH₂O)_(s′), (OCH₂CH₂CH₂)_(s″), (CH₂CH₂CH₂O)_(t), and (aa)_(t′;)

-   aa is independently at each occurrence an amino acid;-   z is selected from the group: aryl substituted with 0-1 R¹⁶, C₃₋₁₀    cycloalkyl substituted with 0-1 R¹⁶, and a 5-10 membered    heterocyclic ring system containing 1-4 heteroatoms independently    selected from N, S, and O and substituted with 0-1 R¹⁶;-   R¹³ R¹⁴, R^(14a), and R¹⁵ are independently selected at each    occurrence from the group: H, ═O, COOH, SO₃H, C₁-C₅ alkyl    substituted with 0-1 R¹⁶, aryl substituted with 0-1 R¹⁶, benzyl    substituted with 0-1 R¹⁶, and C₁-C₅ alkoxy substituted with 0-1 R¹⁶,    NHC(═O)R¹⁷, C(═O)NHR¹⁷, NHC(═O)NHR¹⁷, NHR¹⁷, R¹⁷, and a bond to the    chelator;-   k is 0 or 1;-   s is selected from 0, 1, 2, 3, 4, and 5;-   s′ is selected from 0, 1, 2, 3, 4, and 5;-   s″ is selected from 0, 1, 2, 3, 4, and 5; and-   t is selected from 0, 1, 2, 3, 4, and 5.

(12) A compound according to any one of embodiments 6-11, wherein:

-   W¹ is C(═O)NR¹⁵;-   h is 1;-   g is 3;-   R¹³ and R¹⁴ are independently H;-   x is 1;-   k is 0;-   g′ is 0;-   h′ is 1;-   W² is NH; and-   x′ is 1.

(13) A compound according to any one of embodiments 6-12, wherein:

-   x is 0;-   k is 1;-   Z is aryl substituted with 0-3 R¹⁶;-   g′ is 1;-   W² is NH;-   R^(13a) and R^(14a) are independently H;-   h′ is 1; and-   x′ is 1.

(14) A compound according to any one of embodiments 6-13, wherein:

-   W¹ is C(═O)NR¹⁵;-   h is 1;-   g is 2;-   R¹³ and R¹⁴ are independently H;-   x is 1;-   k is 0;-   g′ is 1;-   R^(13a) and R^(14a) are independently H; or C1-5 alkyl substituted    with 0-3 R¹⁶;-   R¹⁶ is SO₃H;-   W² is NHC(═O) or NH;-   h′ is 1; and-   x′ is 2.

(15) A compound according to any one of embodiments 6-14, wherein:

-   W¹ is C(═O)NH;-   h is 1;-   g is 3;-   R¹³ and R¹⁴ are independently H;-   k is 0;-   g′ is 0;-   x is 1;-   W² is —NH(C═O)— or —(OCH₂CH₂)₇₆₋₈₄-;-   h′ is 2; and-   x′ is 1.

(16) A compound according to any one of embodiments 6-15, wherein:

-   x is 0;-   k is 0;-   g′ is 3;-   h′ is 1;-   W² is NH; and-   x′ is 1.

(17) A compound according to any one of embodiments 6-16, wherein:

-   x is 0;-   Z is aryl substituted with 0-3 R¹⁶;-   k is 1;-   g′ is 1;-   R^(13a)R^(14a) are independently H;-   W² is NHC(═O) or —(OCH2CH2)₇₆₋₈₄-; and-   x′ is 1.

(18) A compound according to any one of embodiments 6-17, wherein:

-   W¹ is C═O;-   g is 2;-   R¹³ and R¹⁴ are independently H;-   k is 0;-   g′ is 0;-   h′ is 1;-   W² is NH; and-   x′ is 1.

(19) A compound according to embodiment 1 wherein the linking group isabsent.

(20) A compound according to any one of embodiments 6-19, wherein thechelator is a metal bonding unit having a formula selected from thegroup:

-   A¹, A², A³, A⁴, A⁵, A⁶, A⁷, and A⁸ are independently selected at    each occurrence from the group: N, NR²⁶,NR¹⁹, NR¹⁹R²⁰, S, SH,    —S(Pg), O, OH, PR¹⁹, PR¹⁹R²⁰, —O—P(O)(R²¹)—O—, P(O)R²¹R²², a bond to    the targeting moiety and a bond to the linking group;-   Pg is a thiol protecting group;-   E¹, E², E³, E⁴, E⁵, E⁶, E⁷, and E⁸ are independently a bond, CH, or    a spacer group independently selected at each occurrence from the    group: C₁-C₁₆ alkyl substituted with 0-3 R²³, aryl substituted with    0-3 R²³, C₃₋₁₀ cycloalkyl substituted with 0-3 R²³,    heterocyclo-C₁₋₁₀ alkyl substituted with 0-3 R²³, wherein the    heterocyclo group is a 5-10 membered heterocyclic ring system    containing 1-4 heteroatoms independently selected from N, S, and O,    C₆₋₁₀ aryl-C₁₋₁₀ alkyl substituted with 0-3 R²³, C₁₋₁₀ alkyl-C₆₋₁₀    aryl-substituted with 0-3 R²³, and a 5-10 membered heterocyclic ring    system containing 1-4 heteroatoms independently selected from N, S,    and O and substituted with 0-3 R²³;-   R¹⁹ and R²⁰ are each independently selected from the group: a bond    to the linking group, a bond to the targeting moiety, hydrogen,    C₁-C₁₀ alkyl substituted with 0-3 R²³, aryl substituted with 0-3    R²³, C₁₋₁₀ cycloalkyl substituted with 0-3 R²³, heterocyclo-C₁₋₁₀    alkyl substituted with 0-3 R²³, wherein the heterocyclo group is a    5-10 membered heterocyclic ring system containing 1-4 heteroatoms    independently selected from N, S, and O, C₆₋₁₀ aryl-C₁₋₁₀ alkyl    substituted with 0-3 R²³, C₁₋₁₀ alkyl-C₆₋₁₀ aryl-substituted with    0-3 R²³, a 5-10 membered heterocyclic ring system containing 1-4    heteroatoms independently selected from N, S, and O and substituted    with 0-3 R²³, and an electron, provided that when one of R¹⁹ or R²⁰    is an electron, then the other is also an electron;-   R²¹ and R²² are each independently selected from the group: a bond    to the linking group, a bond to the targeting moiety, —OH, C₁-C₁₀    alkyl substituted with 0-3 R²³, C₁-C₁₀ alkyl substituted with 0-3    R²³, aryl substituted with 0-3 R²³, C₃₋₁₀ cycloalkyl substituted    with 0-3 R²³, heterocyclo-C₁₋₁₀ alkyl substituted with 0-3 R²³,    wherein the heterocyclo group is a 5-10 membered heterocyclic ring    system containing 1-4 heteroatoms independently selected from N, S,    and O, C₆₋₁₀ aryl-C₁₋₁₀ alkyl substituted with 0-3 R²³, C₁₋₁₀    alkyl-C₆₋₁₀ aryl-substituted with 0-3 R²³, and a 5-10 membered    heterocyclic ring system containing 1-4 heteroatoms independently    selected from N, S, and O and substituted with 0-3 R²³;-   R²³ is independently selected at each occurrence from the group:    -   a bond to the linking group, a bond to the targeting moiety, ═O,        F, Cl, Br, I, —CF₃, —CN, —CO₂R²⁴, —C(═O)R²⁴, —C(═O)N(R²⁴)₂,        —CHO, —CH₂OR²⁴, —OC(═O)R²⁴, —OC(═O)OR^(24a), —OR²⁴,        —OC(═O)N(R²⁴)₂, —NR²⁵C(═O)R²⁴, —NR²⁵C(═O)OR^(24a),        —NR²⁵C(═O)N(R²⁴)₂, —NR²⁵SO₂N(R²⁴)₂, —NR²⁵SO₂R^(24a), —SO₃H,        SO₂R^(24a), —SR²⁴, —S(═O)R^(24a), —SO₂N(R²⁴)₂, —N(R²⁴)₂,        —NHC(═S)NHR²⁴, ═NOR²⁴, NO₂, —C(═O)NHOR²⁴, —C(═O)NHNR²⁴R^(24a),        —OCH₂CO₂H, 2-(1-morpholino)ethoxy, C₁-C₅ alkyl, C₂-C₄ alkenyl,        C₃-C₆ cycloalkyl, C₃-C₆ cycloalkylmethyl, C₂-C₆ alkoxyalkyl,        aryl substituted with 0-2 R²⁴, and a 5-10 membered heterocyclic        ring system containing 1-4 heteroatoms independently selected        from N, S, and O; and-   wherein at least one of A¹, A², A³, A⁴, A⁵, A⁶, A⁷, A⁸ or R²³ is a    bond to the linking group or targeting moiety;-   R²⁴, R^(24a), and R²⁵ are independently selected at each occurrence    from the group: a bond to the linking group, a bond to the targeting    moiety, H, C₁-C₆ alkyl, phenyl, benzyl, C₁-C₆ alkoxy, halide, nitro,    cyano, and trifluoromethyl; and-   R²⁶ is a co-ordinate bond to a metal or a hydrazine protecting    group.

(21) A compound according to any one of embodiments 6-20 wherein:

-   A¹, A², A³, A⁴, A⁵, A⁶, A⁷, and A⁸ are independently selected at    each occurrence from the group: NR¹ ⁹, NR¹⁹R²⁰, S, SH, OH, a bond to    the targeting moiety and a bond to the linking group;-   E¹, E², E³, E⁴, E⁵, E⁶, E⁷, and E⁸ are independently a bond, CH, or    a spacer group independently selected at each occurrence from the    group: C₁-C₁₀ alkyl substituted with 0-3 R²³, aryl substituted with    0-3 R²³, C₃₋₁₀ cycloalkyl substituted with 0-3 R²³, and a 5-10    membered heterocyclic ring system containing 1-4 heteroatoms    independently selected from N, S, and O and substituted with 0-3    R²³;-   wherein at least one of A¹, A², A³, A⁴, A⁵, A⁶, A⁷, A⁸ and R²³ is a    bond to the linking group or a targeting moiety;-   R¹⁹, and R²⁰ are each independently selected from the group: a bond    to the targeting moiety, a bond to the linking group, hydrogen,    C₁-C₁₀ alkyl substituted with 0-3 R²³, aryl substituted with 0-3    R²³, a 5-10 membered heterocyclic ring system containing 1-4    heteroatoms independently selected from N, S, and O and substituted    with 0-3 R²³, and an electron, provided that when one of R¹⁹ or R²⁰    is an electron, then the other is also an electron;-   R²³ is independently selected at each occurrence from the group: a    bond to the targeting moiety, a bond to the linking group, ═O, F,    Cl, Br, I, —CF₃, —CN, —CO₂R²⁴, —C(═O)R²⁴, —C(═O)N(R²⁴)₂, —CH₂OR²⁴,    —OC(═O)R²⁴, —OC(═O)OR^(24a), —OR²⁴, —OC(═O)N(R²⁴)₂, —NR²⁵C(═O)R²⁴,    —NR²⁵C(═O)OR^(24a), —NR²⁵C(═O)N(R²⁴)₂, —NR²⁵SO₂N(R²⁴)₂,    —NR²⁵SO₂R^(24a), —SO₃H, —SO₂R^(24a), —S(═O)R^(24a), —SO₂N(R²⁴)₂,    —N(R²⁴)₂, —NHC(═S)NHR²⁴, ═NOR¹⁸, —C(═O)NHNR¹⁸R^(18a), —OCH₂CO₂H, and    2-(1-morpholino)ethoxy; and-   R²⁴, R^(24a), and R²⁵ are independently selected at each occurrence    from the group: a bond to the linking group, H, and C₁-C₆ alkyl.

(22) A compound according to any one of embodiments 6-21 wherein thechelator is of the formula:

-   A¹ is a bond to the linking group;-   A², A⁴, and A⁶ are each N;-   A³, A⁵, A⁷ and A⁸ are each OH;-   E¹, E², and E⁴ are C2 alkyl;-   E³, E⁵, E⁷, and E⁸ are C2 alkyl substituted with 0-1 R²³;-   R²³ is ═O;

(23) A compound according to any one of embodiments 6-22 wherein thechelator is of the formula:

-   Ch is    wherein:-   A5 is a bond to Ln;-   A¹, A³, A⁷ and A⁸ are each OH;-   A², A⁴ and A⁶ are each NH;-   E¹, E³, E⁵, E⁷, and E⁸ are C2 alkyl substituted with 0-1 R²³;-   E², and E⁴, are C2 alkyl;-   R²³ is ═O

(24) A compound according to any one of embodiments 6-23 wherein thechelator is of the formula:

-   A¹, A², A³ and A⁴ are each N;-   A⁵, A⁶ and A⁸ are each OH;-   A⁷ is a bond to L_(n);-   E¹, E², E³, E⁴ are each independently C₂ alkyl; and-   E⁵, E⁶, E⁷, E⁸ are each independently C₂ alkyl substituted with 0-1    R²³;-   R²³ is ═O.

(25) A compound according to any one of embodiments 6-24 wherein thechelator is of the formula:

-   A¹ is NR²⁶;-   R²⁶ is a co-ordinate bond to a metal or a hydrazine protecting    group;-   E¹ is a bond;-   A² is NHR19;-   R¹⁹ is a heterocycle substituted with R²³, the heterocycle being    selected from pyridine and pyrimidine;-   R²³ is selected from a bond to the linking group, C(═O)NHR²⁴ and    C(═O)R²⁴; and-   R²⁴ is a bond to the linking group.

(26) A compound according to any one of embodiments 6-25 wherein thechelator is of the formula:

wherein:

-   A¹ and A⁵ are each —S(Pg);-   Pg is a thiol protecting group;-   E¹ and E⁴ are C₂ alkyl substituted with 0-1 R²³;-   R²³ is ═O;-   A² and A⁴ are each —NH;-   E² is CH₂;-   E³ is C₁₋₃ alkyl substituted with 0-1 R²³;-   A³ is a bond to Ln.

(27) A compound according to any one of embodiments 6-26 wherein thechelator is of the formula:

wherein:

-   A¹ is a bond to Ln;-   E¹ is C₁ alkyl substituted by R²³;-   A² is NH;-   E² is C₂ alkyl substituted with 0-1R²³;-   A³ is —O—P(O)(R²¹) —O;-   E³ is C₁ alkyl;-   A⁴ and A⁵ are each —O—;-   E⁴ and E ⁶ are each independently C₁₋₁₆ alkyl substituted with    1-1R²³;-   E⁵ C₁ alky-   R²¹ is —OH; and-   R²³ is ═O.

(28) A compound of embodiment 1 having the formula:(Q)_(d)—L_(n)—C_(h)wherein, Q is a compound of Formulae (Ia) or (Ib):

wherein,

-   R is independently OH or —CH₂SH;-   R¹ is independently selected at each occurrence from the group: H,    OH, C₁₋₃ alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl, and heterocycle-S—CH₂—;-   R² is independently C₁₋₂₀ alkyl;-   X is independently C═O or SO₂, provided when X is C═O, R³ is    and when X is SO₂, R³ is independently selected from the group: aryl    substituted with 0-2 R⁶, and heterocycle substituted with 0-2 R⁶;-   R⁴ is independently selected at each occurrence from the group: C₁₋₆    alkyl, phenyl, and benzyl;-   R⁵ is independently at each occurrence from the group: NH(C₁₋₆    alkyl), NH-phenyl, and NH-heterocycle; wherein said alkyl, phenyl    and heterocycle groups are optionally substituted with a bond to    L_(n);-   R⁶ is independently aryloxy substituted with 0-3 R⁷;-   R⁷ is independently halogen or methoxy;    or alternatively,-   R¹ and R⁴ may be taken together to form a bridging group of the    formula —(CH₂)₃—O-phenyl-CH₂—, optionally substituted with a bond to    L_(n);    or alternatively,-   R¹ and R² may be taken together to form a bridging group of the    formula —(CH₂)₃—NH—, optionally substituted with a bond to L_(n); or-   R¹ and R² taken together with the nitrogen and carbon atom through    which they are attached form a C₅₋₇ atom saturated ring system    substituted with one or more substituents selected from the group    consisting of: a bond to Ln, a bond to C_(h), and —C(═O)—NR²⁹R³⁰;-   R⁸ is independently at each occurrence OH or phenyl, optionally    substituted with a bond to L_(n), provided that when R⁸ is phenyl,    R¹⁰ is —C(═O)—CR¹²—NH—CH(CH₃)—COOH;-   R⁹ and R⁹′ are independently H, C₁₋₆ alkyl optionally substituted    with a bond to L_(n), or are taken together with the carbon atom to    which they are attached to form a 5-7 atom saturated, partially    unsaturated or aromatic ring system containing 0-3 heteroatoms    selected from O, N, SO₂ and S, said ring system substituted with R⁶    and optionally substituted with a bond to L_(n);-   R¹⁰ and R¹¹ are independently H, or C₁₋₆ alkyl optionally    substituted with a bond to L_(n), or are taken together with the    nitrogen atom to which they are attached to form a 5-7 atom    saturated, partially unsaturated or aromatic ring system containing    0-3 heteroatoms selected from O, N, SO₂ and S, said ring system    optionally substituted with 0-3 R²⁷ or a bond to L_(n);    or alternatively,-   R⁹ and R¹⁰ are taken together with the carbon atom to which they are    attached to form a 5-7 atom saturated, partially unsaturated or    aromatic ring system containing 0-3 heteroatoms selected from O, N,    SO₂ and S, said ring system optionally substituted with a bond to    L_(n);-   R¹² is independently C₁₋₂₀ alkyl;-   d is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   L_(n) is a linking group having the formula:    ((W¹)_(h)—(CR¹³R¹⁴)_(g))_(x)—(Z)_(k)—((CR^(13a)R^(14a))_(g′)—(Ser.    No. W²)_(h′)) _(x′;)-   W¹ and W² are independently selected at each occurrence from the    group: O, S, NH, NHC(═O), C(═O)NH, NR¹⁵C(═O), C(═O)NR¹⁵, C(═O),    C(═O)O, OC(═O), NHC(═S)NH, NHC(═O)NH, SO₂, SO₂NH, —(OCH₂CH₂)₇₆₋₈₄,    (OCH₂CH₂)_(s), (CH₂CH₂O)_(s′), (OCH₂CH₂CH₂)_(s″), (CH₂CH₂CH₂O)_(t),    and (aa)_(t′;)-   aa is independently at each occurrence an amino acid;-   Z is selected from the group: aryl substituted with 0-3 R¹⁶, C₃₋₁₀    cycloalkyl substituted with 0-3 R¹⁶, and a 5-10 membered    heterocyclic ring system containing 1-4 heteroatoms independently    selected from N, S, and O and substituted with 0-3 R¹⁶;-   R¹³, R¹⁴, R^(14a), and R¹⁵ are independently selected at each    occurrence from the group: H, ═O, COOH, SO₃H, PO₃H, C₁-C₅ alkyl    substituted with 0-3 R¹⁶, aryl substituted with 0-3 R¹⁶, benzyl    substituted with 0-3 R¹⁶, and C₁-C₅ alkoxy substituted with 0-3 R¹⁶,    NHC(═O)R¹⁷, C(—O)NHR¹⁷, NHC(═O)NHR¹⁷, NHR¹⁷, R¹⁷, and a bond to    C_(h);-   R¹⁶ is independently selected at each occurrence from the group: a    bond to C_(h), COOR¹⁷, C(═O)NHR¹⁷, NHC(═O)R¹⁷, OH, NHR¹⁷, SO₃H,    PO₃H, —OPO₃H₂, —OSO₃H, aryl substituted with 0-3 R¹⁷, C₁₋₅ alkyl    substituted with 0-1 R¹⁸, C₁₋₅ alkoxy substituted with 0-1 R¹⁸, and    a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms    independently selected from N, S, and O and substituted with 0-3    R¹⁷;-   R¹⁷ is independently selected at each occurrence from the group: H,    alkyl substituted with 0-1 R¹⁸, aryl substituted with 0-1 R¹⁸, a    5-10 membered heterocyclic ring system containing 1-4 heteroatoms    independently selected from N, S, and O and substituted with 0-1    R¹⁸, C₃₋₁₀ cycloalkyl substituted with 0-1 R¹⁸, polyalkylene glycol    substituted with 0-1 R¹⁸, carbohydrate substituted with 0-1 R¹⁸,    cyclodextrin substituted with 0-1 R¹⁸, amino acid substituted with    0-1 R¹⁸, polycarboxyalkyl substituted with 0-1 R¹⁸, polyazaalkyl    substituted with 0-1 R¹⁸, peptide substituted with 0-1 R¹⁸, wherein    the peptide is comprised of 2-10 amino acids,    3,6-O-disulfo-B-D-galactopyranosyl, bis(phosphonomethyl)glycine, and    a bond to C_(h);-   R¹⁸ is a bond to C_(h);-   k is selected from 0, 1, and 2;-   h is selected from 0, 1, and 2;-   h′ is selected from 0, 1, and 2;-   g is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   g′ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   s is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   s′ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   s″ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   t is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   t′ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   x is selected from 0, 1, 2, 3, 4, and 5;-   x′ is selected from 0, 1, 2, 3, 4, and 5;-   C_(h) is a metal bonding unit having a formula selected from the    group:

A¹, A², A³, A⁴, A⁵, A⁶, A⁷, and A⁸ are independently selected at eachoccurrence from the group: N, NR²⁶,NR¹⁹, NR¹⁹R²⁰, S, SH, —S(Pg), O, OH,PR¹⁹, PR¹⁹R²⁰, —O—P(O)(R²¹)—O—, P(O)R²¹R²², a bond to the targetingmoiety and a bond to the linking group;

-   Pg is a thiol protecting group;-   E¹, E², E³, E⁴, E⁵, E⁶, E⁷, and E⁸ are independently a bond, CH, or    a spacer group independently selected at each occurrence from the    group: C₁-C₁₆ alkyl substituted with 0-3 R²³, aryl substituted with    0-3 R²³, C₃₋₁₀ cycloalkyl substituted with 0-3 R²³,    heterocyclo-C₁₋₁₀ alkyl substituted with 0-3 R²³, wherein the    heterocyclo group is a 5-10 membered heterocyclic ring system    containing 1-4 heteroatoms independently selected from N, S, and O,    C₆₋₁₀ aryl-C₁₋₁₀ alkyl substituted with 0-3 R²³, C₁₋₁₀ alkyl-C₆₋₁₀    aryl-substituted with 0-3 R²³, and a 5-10 membered heterocyclic ring    system containing 1-4 heteroatoms independently selected from N, S,    and O and substituted with 0-3 R²³;-   R¹⁹ and R²⁰ are each independently selected from the group: a bond    to the linking group, a bond to the targeting moiety, hydrogen,    C₁-C₁₀ alkyl substituted with 0-3 R²³, aryl substituted with 0-3    R²³, C₁₋₁₀ cycloalkyl substituted with 0-3 R²³, heterocyclo-C₁₋₁₀    alkyl substituted with 0-3 R²³, wherein the heterocyclo group is a    5-10 membered heterocyclic ring system containing 1-4 heteroatoms    independently selected from N, S, and O, C₆₋₁₀ aryl-C₁₋₁₀ alkyl    substituted with 0-3 R²³, C₁₋₁₀ alkyl-C₆₋₁₀ aryl-substituted with    0-3 R²³, a 5-10 membered heterocyclic ring system containing 1-4    heteroatoms independently selected from N, S, and O and substituted    with 0-3 R²³, and an electron, provided that when one of R¹⁹ or R²⁰    is an electron, then the other is also an electron;-   R²¹ and R²² are each independently selected from the group: a bond    to the linking group, a bond to the targeting moiety, —OH, C₁-C₁₀    alkyl substituted with 0-3 R²³, C₁-C₁₀ alkyl substituted with 0-3    R²³, aryl substituted with 0-3 R²³, C₃₋₁₀ cycloalkyl substituted    with 0-3 R²³, heterocyclo-C₁₋₁₀ alkyl substituted with 0-3 R²³,    wherein the heterocyclo group is a 5-10 membered heterocyclic ring    system containing 1-4 heteroatoms independently selected from N, S,    and O, C₆₋₁₀ aryl-C₁₋₁₀ alkyl substituted with 0-3 R²³, C₁₋₁₀    alkyl-C₆₋₁₀ aryl substituted with 0-3 R²³, and a 5-10 membered    heterocyclic ring system containing 1-4 heteroatoms independently    selected from N, S, and O and substituted with 0-3 R²³;-   R²³ is independently selected at each occurrence from the group: a    bond to the linking group, a bond to the targeting moiety, ═O, F,    Cl, Br, I, —CF₃, —CN, —CO₂R²⁴, —C(═O)R²⁴, —C(═O)N(R²⁴)₂, —CHO,    —CH₂OR²⁴, —OC(═O)R²⁴, —OC(═O)OR^(24a), —OR²⁴, —OC(═O)N(R²⁴)₂,    —NR²⁵C(═O)R²⁴, —NR²⁵C(═O)OR^(24a), —NR²⁵C(═O)N(R²⁴)₂,    —NR²⁵SO₂N(R²⁴)₂, —NR²⁵SO₂R^(24a), —SO₃H, —SO₂R^(24a), —SR²⁴,    —S(═O)R^(24a), —SO₂N(R²⁴)₂, —N(R²⁴)₂, —NHC(═S)NHR²⁴, ═NOR²⁴, NO₂,    —C(═O)NHOR²⁴, —C(═O)NHNR²⁴R^(24a), —OCH₂CO₂H,    2-(1-morpholino)ethoxy, C₁-C₅ alkyl, C₂-C₄ alkenyl, C₃-C₆    cycloalkyl, C₃-C₆ cycloalkylmethyl, C₂-C₆ alkoxyalkyl, aryl    substituted with 0-2 R²⁴, and a 5-10 membered heterocyclic ring    system containing 1-4 heteroatoms independently selected from N, S,    and O; and-   wherein at least one of A¹, A², A³, A⁴, A⁵, A⁶, A⁷, A⁸ or R²³ is a    bond to the linking group or targeting moiety;-   R²⁴, R^(24a), and R²⁵ are independently selected at each occurrence    from the group: a bond to the linking group, a bond to the targeting    moiety, H, C₁-C₆ alkyl, phenyl, benzyl, C₁-C₆ alkoxy, halide, nitro,    cyano, and trifluoromethyl; and-   R²⁶ is a co-ordinate bond to a metal or a hydrazine protecting    group; or-   a pharmaceutically acceptable salt thereof.

(29) A compound according to embodiment 28 wherein:

-   R is —OH;-   R² is C1-6 alkyl;-   X is C═O;-   R³ is

R¹ and R⁴ are taken together to form a bridging group of formula—(CH₂)₃—O-phenyl-CH₂—;

-   R⁵ is NH(C1-6alkyl), substituted with a bond to the linking group or    a bond to the chelator.

(30) A compound according to any one of embodiments 28-29 wherein:

-   R is —OH;-   R⁹ is C₁ alkyl substituted with a bond to Ln;-   R¹⁰ and R¹¹ taken together with the nitrogen atom to which they are    attached form a 5 atom saturated ring system, said right system is    substituted with 0-3 R²⁷;-   R²⁷ is ═O, C1-4 alkyl, or phenyl substituted with R²⁸; and-   R²⁸ is a phenoxy group substituted with 0-2 OCH₃ groups.

(31) A compound according to any one of embodiments 28-30 wherein

-   R is —OH;-   R¹ and R² taken together with the nitrogen and carbon atom through    which they are attached form a C₅₋₇ atom saturated ring system    substituted with one or more substituents selected from the group    consisting of: a bond to Ln, a bond to C_(h), and —C(═O)—NR²⁹R³⁰;-   R²⁹ and R³⁰ taken together with the nitrogen atom through which they    are attached form a C₅₋₇ atom saturated ring system substituted with    R³¹; and-   R³¹ is a benzyloxy group substituted with C1-4 alkyl.

(32) A compound according to any one of embodiments 28-31 wherein

-   d is selected from 1, 2, 3, 4, and 5;-   W is independently selected at each occurrence from the group: O,    NH, NHC(═O), C(═O)NH, NR¹⁵C(═O), C(═O)NR¹⁵, C(═O), C(═O)O, OC(═O),    NHC(═S)NH, NHC(═O)NH, SO₂, (OCH₂CH₂)s, (CH₂CH₂O)_(s′),    (OCH₂CH₂CH₂)_(s″), (CH₂CH₂CH₂O)_(t), and (aa)_(t′;)-   aa is independently at each occurrence an amino acid;-   Z is selected from the group: aryl substituted with 0-1 R¹⁶, C₃₋₁₀    cycloalkyl substituted with 0-1 R¹⁶, and a 5-10 membered    heterocyclic ring system containing 1-4 heteroatoms independently    selected from N, S, and O and substituted with 0-1 R¹⁶;-   R¹³, R^(13a), R¹⁴, R^(14a), and R¹⁵ are independently selected at    each occurrence from the group: H, ═O, COOH, SO₃H, C₁-C₅ alkyl    substituted with 0-1 R¹⁶, aryl substituted with 0-1 R¹⁶, benzyl    substituted with 0-1 R¹⁶, and C₁-C₅ alkoxy substituted with 0-1 R¹⁶,    NHC(═O)R¹⁷, C(═O)NHR¹⁷, NHC(═O)NHR¹⁷, NHR¹⁷, R¹⁷, and a bond to    C_(h);-   k is 0 or 1;-   is selected from 0, 1, 2, 3, 4, and 5;-   s′ is selected from 0, 1, 2, 3, 4, and 5;-   s″ is selected from 0, 1, 2, 3, 4, and 5;-   t is selected from 0, 1, 2, 3, 4, and 5;-   A¹, A², A³, A⁴, A⁵, A⁶, A⁷, and A⁸ are independently selected at    each occurrence from the group: NR¹⁹, NR¹⁹R²⁰, S, SH, OH, and a bond    to L_(n);-   E is a bond, CH, or a spacer group independently selected at each    occurrence from the group: C₁-C₁₀ alkyl substituted with 0-3 R²³,    aryl substituted with 0-3 R²³, C₃₋₁₀ cycloalkyl substituted with 0-3    R²³, and a 5-10 membered heterocyclic ring system containing 1-4    heteroatoms independently selected from N, S, and O and substituted    with 0-3 R²³;-   R¹⁹, and R²⁰ are each independently selected from the group: a bond    to L_(n), hydrogen, C₁-C₁₀ alkyl substituted with 0-3 R²³, aryl    substituted with 0-3 R²³, a 5-10 membered heterocyclic ring system    containing 1-4 heteroatoms independently selected from N, S, and O    and substituted with 0-3 R²³, and an electron, provided that when    one of R¹⁹ or R²⁰ is an electron, then the other is also an    electron;-   R²³ is independently selected at each occurrence from the group: a    bond to L_(n), ═O, F, Cl, Br, I, —CF₃, —CN, —CO₂R²⁴—C(═O)R²⁴,    —C(═O)N(R²⁴)₂, —CH₂OR²⁴, —OC(═O)R²⁴, —OC(═O)OR^(24a), —OR²⁴,    —OC(═O)N(R²⁴)₂, —NR²⁵C(═O)R²⁴, —NR²⁵C(═O)OR^(24a),    —NR²⁵C(═O)N(R²⁴)₂, —NR²⁵SO₂N(R²⁴)₂, —NR²SO₂R^(24a), —SO₃H,    —SO₂R^(24a), —S(═O)R^(24a), —SO₂N(R²⁴)₂, —N(R²⁴)₂, —NHC(═S)NHR²⁴,    ═NOR¹⁸, —C(═O)NHNR¹⁸R^(18a), —OCH₂CO₂H, and 2-(1-morpholino)ethoxy;    and-   R²⁴, R^(24a), and R²⁵ are independently selected at each occurrence    from the group: a bond to L_(n), H, and C₁-C₆ alkyl; and

(33) A compound according to any one of embodiments 28-32 wherein

-   d is 1,-   C_(h) is-   A¹ is a bond to L_(n);-   A², A⁴, and A⁶ are each N;-   A³, A⁵, A⁷ and A⁸ are each OH;-   E¹, E², and E⁴ are C2 alkyl;-   E³, E⁵, E⁷, and E⁸ are C₂ alkyl substituted with 0-1 R²³;-   R²³ is ═O;

(34) A compound according to any one of embodiments 28-33 wherein

-   C_(h) is    wherein:-   A5 is a bond to Ln;-   A¹, A³, A⁷ and A⁸ are each OH;-   A², A⁴ and A⁶ are each NH;-   E¹E³, E⁵, E⁷, and E⁸ are C₂ alkyl substituted with 0-1 R²³;-   E², and E⁴, are C₂ alkyl;-   R²³ is ═O.

(35) A compound according to any one of embodiments 28-34 is wherein

-   C_(h) is-   A¹, A², A³ and A⁴ are each N;-   A⁵, A⁶ and A⁸ are each OH;-   A⁷ is a bond to L_(n);-   E¹, E², E³, E⁴ are each independently, C₂ alkyl; and-   E⁵, E⁶, E⁷, E⁸ are each independently, C₂ alkyl substituted with 0-1    R²³;-   R²³ is ═O;

(36) A compound according to any one of embodiments 28-35 wherein

-   C_(h) is A¹-   A¹ is NR²⁶;-   R²⁶ is a co-ordinate bond to a metal; or a hydrazine protecting    group;-   E¹ is a bond;-   A² is NHR¹⁹;-   R¹⁹ is a heterocycle substituted with R²³, the heterocycle being    selected from pyridine and pyrimidine;-   R²³ is selected from a bond to L_(n), C(═O)NHR²⁴ and C(═O)R²⁴; and-   R²⁴ is a bond to L_(n).

(37) A compound according to any one of embodiments 28-36 wherein

wherein:

-   A¹ and A⁵ are each —S(Pg);-   Pg is a thiol protecting group;-   E¹ and E⁴ are C₂ alkyl substituted with 0-1 R²³;-   R²³ is =O;-   A² and A⁴ are each —NH;-   E² is CH₂;-   E³ is C₁₋₃ alkyl substituted with 0-1 R²³;-   A³ is a bond to Ln.

(38) A compound according to any one of embodiments 28-37 wherein

wherein:

-   A¹ ia a bond to Ln;-   E¹ is C₁ alkyl substituted by R²³;-   A² is NH;-   E² is C₂ alkyl sunsttuted wth 0-1R²³;-   A³ is —O—P(O) (R²¹)—O;-   E³ is C₁ alkyl;-   A⁴ and A⁵ are each —O—;-   E⁴ and E⁶ are each independently C₁₋₆ alkyl substituted with 0-1R²³;-   E⁵ is C₁ alkyl;-   A⁵ is —O—;-   R²¹ is —OH; and-   R²³ is ═O

(39) A compound according embodiment 28 wherein

-   W¹ is C(═O)NR¹⁵;-   h is 1;-   g is 3;-   R¹³ and R¹⁴ are independently H;-   x is 1;-   k is 0;-   g′ is 0;-   h′ is 1;-   W² is NH; and-   x′ is 1

(40) A compound according to embodiments 28 wherein

-   x is 0;-   k is 1;-   Z is aryl substituted with 0-3 R¹⁶;-   g′ is 1;-   W² is NH;-   R^(13a)and R^(14a)are independently H;-   h′ is 1; and-   x′ is 1.

(41) A compound according to embodiments 28 wherein

-   W¹ is C(═O)NR¹⁵;-   h is 1;-   g is 2;-   R¹³ and R¹⁴ are independently H;-   x is 1;-   k is 0;-   g′ is 1;-   R^(13a) and R^(14a) are independently H; or C1-5 alkyl substituted    with 0-3 R¹⁶;-   R¹⁶ is SO₃H;-   W² is NHC(═O) or NH;-   h′ is 1; and-   x′ is 2.

(42) A compound according to embodiment 28 wherein

-   W¹ is C(═O)NH;-   h is 1;-   g is 3;-   R¹³ and R¹⁴ are independently H;-   k is 0;-   g′ is 0;-   x is 1;-   W² is —NH(C═O)— or —(OCH₂CH₂)₇₆₋₈₄-;-   h′ is 2; and-   x′ is 1.

(43) A compound according to embodiment 28 wherein

-   x is 0;-   k is 0;-   g′ is 3;-   h′ is 1;-   W² is NH; and-   x′ is 1.

(44) A compound according to embodiment 28 wherein

-   x is 0;-   Z is aryl substituted with 0-3 R¹⁶;-   k is 1;-   g′ is 1;-   R^(13a) R^(14a) are independently H;-   W² is NHC(═O) or —(OCH2CH2)₇₆₋₈₄-; and-   x′ is 1.

(45) A compound according to embodiment 28 wherein

-   W¹ is C═O;-   g is 2;-   R¹³ and R¹⁴ are independently H;-   k is 0;-   g′ is 0;-   h′ is 1;-   W² is NH; and-   x′ is 1.

(46) A compound according to embodiment 1 or 28 selected from the groupconsisting of:2-{[5-(3-{2-[(6-Hydroxycarbamoyl-7-isobutyl-8-oxo-2-oxa-9-aza-bicyclo[10.2.2]hexadeca-1(15),12(16),13-triene-10-carbonyl)-amino]-acetylamino}-propylcarbamoyl)-pyridin-2-yl]-hydrazonomethyl}-benzenesulfonicacid;

-   2-{[5-(4-{[(6-Hydroxycarbamoyl-7-isobutyl-8-oxo-2-oxa-9-aza-bicyclo[10.2.2]hexadeca-1(15),12(16),13-triene-10-carbonyl)-amino]-methyl}-benzylcarbamoyl)-pyridin-2-yl]-hydrazonomethyl)-benzenesulfonic    acid;-   2-[7-((N-[3-(2-{[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]carbonylamino}acetylamino)propyl]carbamoyl)methyl)-1,4,7,10-tetraaza-4,10-bis(carboxymethyl)cyclododecyl]acetic    acid;-   2-{7-[(N-{[4-({[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]-carbonylamino}methyl)phenyl]methyl}carbamoyl)methyl]-1,4,7,10-tetraaza-4,10-bis(carboxymethyl)cyclododecyl]acetic    acid;-   2-(7-{[N-(1-{N-[3-(2-{[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-ll-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]carbonylamino}acetylamino)propyl]carbamoyl}-2-sulfoethyl)carbamoyl]methyl)-1,4,7,10-tetraaza-4,10-bis(carboxymethyl)cyclododecyl)acetic    acid;-   2-[7-({N-[1-(N-{[4-({[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]-carbonylamino)methyl)phenyl]methyl]carbamoyl]-2-sulfoethyl]carbamoyl)methyl)-1,4,7,10-tetraaza-4,10-bis(carboxymethyl)cyclododecyl]acetic    acid;-   2-({2-[({N-[3-(2-{[7(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]carbonylamino}acetylamino)propyl]carbamoyl}methyl)(carboxymet    hyl)amino)ethyl){2-[bis(carboxymethyl)amino]ethyl}amino]acetic acid;-   2-[(2-{[(N-{[4-({[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]-carbonylamino}methyl)phenyl]methyl}carbamoyl)methyl](carboxymeth    yl)amino}ethyl){2-[bis(carboxymethyl)amino]ethyl}amino]acetic acid;-   N-[3-(2-{[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]carbonylamino}acetylamino)propyl]-4,5-bis[2-(ethoxyethylthio)acetylamino]pentanamide;-   N-{[4-({[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]carbonylamino}methyl)-phenyl]methyl}-4,5-bis[2-(ethoxyethylthio)acetylamino]-pentanamide;-   1-(1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamino)-α,    ω-dicarbonylPEG₃₄₀₀-2-{[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]carbonylamino}-N-(3-aminopropyl)acetamide;-   1-(1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamino)-α,    ω-dicarbonylPEG₃₄₀₀-[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]-N-{[4-(aminomethyl)phenyl]methyl}carboxamide    conjugate;-   2-[2-({5-[N-(5-(N-hydroxycarbamoyl)(5R)-5-{3-[4-(3,4-dimethoxyphenoxy)phenyl]-3-methyl-2-oxopyrrolidinyl}pentyl)carbamoyl](2-pyridyl)}amino)(1Z)-2-azavinyl]benzenesulfonic    acid;-   2-(2-{[5-(N-{3-[3-(N-hydroxycarbamoyl)(4S)-4-(}4-[(4-methylphenyl)methoxy]piperidyl]carbonyl)piperidyl]-3-oxopropyl}carbamoyl)(2-pyridyl)]amino}(1Z)-2-azavinyl)benzenesulfonic    acid; and

(47) A radiopharmaceutical comprising a compound of any one ofembodiments 1-46 and a cytotoxic radioisotope which is complexed to thechelator.

(48) A radiopharmaceutical comprising a of any one of embodiments 1-47and a cytotoxic radioisotope which is complexed to the chelator.

(49) A radiopharmaceutical comprising a compound of any one ofembodiments 1-47 and a cytotoxic radioisotope.

(50) A radiopharmaceutical according to embodiment 20 selected from thegroup consisting of:

-   2-{[5-(3-{2-[(6-Hydroxycarbamoyl-7-isobutyl-8-oxo-2-oxa-9-aza-bicyclo[10.2.2]hexadeca-1(15),12(16),13-triene-10-carbonyl)-amino]-acetylamino}-propylcarbamoyl)-pyridin-2-yl]-hydrazonomethyl}-benzenesulfonic    acid; and-   2-{[5-(4-{[(6-Hydroxycarbamoyl-7-isobutyl-8-oxo-2-oxa-9-aza-bicyclo[10.2.2]hexadeca-1(15),12(16),13-triene-10-carbonyl)-amino]-methyl}-benzylcarbamoyl)-pyridin-2-yl]-hydrazonomethyl}-benzenesulfonic    acid;    wherein the cytotoxic radioisotope is ^(99m)Tc.

(51) A radiopharmaceutical according to embodiment 47 wherein thecytotoxic radioisotope is selected from the group consisting of betaparticle emitters, alpha particle emitters, and Auger electron emitters.

(52) A radiopharmaceutical according to embodiment 47 wherein thecytotoxic radioisotope is selected from the group consisting of: ¹⁸⁶Re,¹⁸⁸Re, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁴⁹ Pm, ⁹⁰Y, ²¹²Bi, ¹⁰³Pd, ¹⁰⁹Pd, ¹⁵⁹Gd,¹⁴⁰La, ¹⁹⁸Au, ¹⁹⁹Au, 169Yb, ¹⁷⁵Yb, 165Dy, 166Dy, ⁶⁷Cu, ¹⁰⁵Rh, ¹¹¹Ag, and¹⁹²Ir.

(53) A radiopharmaceutical according to embodiment 47 wherein thecytotoxic radioisotope is selected from the group consisting of: ¹⁸⁶Re,¹⁸⁸Re, 153Sm, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁴⁹ Pm, ⁹⁰Y, ²¹²Bi, ¹⁰³Pd, and 105Rh.

(54) A radiopharmaceutical according to embodiment 47 wherein thecytotoxic radioisotope is selected from the group consisting of: ¹⁸⁶Re,¹⁸⁸Re, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁴⁹ Pm, ⁹⁰Y, and ²¹²Bi.

(55) A composition comprising a compound of any one of embodiments 1-54,or a pharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier.

(56) A radiopharmaceutical composition comprising a compound of any oneof embodiments 1-55, or a pharmaceutically acceptable salt thereof, anda pharmaceutically acceptable carrier.

(57) A radiopharmaceutical composition according to embodiment 56,further comprising at least one agent selected from the group consistingof a chemotherapeutic agent and a radiosensitizer agent, or apharmaceutically acceptable salt thereof.

(58) A radiopharmaceutical composition according to embodiment 57,wherein the chemotherapeutic agent is selected from the group consistingof mitomycin, tretinoin, ribomustin, gemcitabine, vincristine,etoposide, cladribine, mitobronitol, methotrexate, doxorubicin,carboquone, pentostatin, nitracrine, zinostatin, cetrorelix, letrozole,raltitrexed, daunorubicin, fadrozole, fotemustine, thymalfasin,sobuzoxane, nedaplatin, cytarabine, bicalutamide, vinorelbine,vesnarinone, aminoglutethimide, amsacrine, proglumide, elliptiniumacetate, ketanserin, doxifluridine, etretinate, isotretinoin,streptozocin, nimustine, vindesine, flutamide, drogenil, butocin,carmofur, razoxane, sizofilan, carboplatin, mitolactol, tegafur,ifosfamide, prednimustine, picibanil, levamisole, teniposide,improsulfan, enocitabine, lisuride, oxymetholone, tamoxifen,progesterone, mepitiostane, epitiostanol, formestane, interferon-alpha,interferon-2 alpha, interferon-beta, interferon-gamma, colonystimulating factor-1, colony stimulating factor-2, denileukin diftitox,interleukin-2, and leutinizing hormone releasing factor.

(59) A radiopharmaceutical composition according to embodiment57,wherein radiosensitizer agent is selected from the group consiting of2-(3-nitro-1,2,4-triazol-1-yl)-N-(2-methoxyethyl)acetamide,N-(3-nitro-4-quinolinyl)-4-morpholinecarboxamidine,3-amino-1,2,4-benzotriazine-1,4-dioxide,N-(2-hydroxyethyl)-2-nitroimidazole-1-acetamide,1-(2-nitroimidazol-1-yl)-3-(1-piperidinyl)-2-propanol, and1-(2-nitro-1-imidazolyl)-3-(1-aziridino)-2-propanol.

(60) A kit comprising a compound of Embodiment 1, or a pharmaceuticallyacceptable salt form thereof and a pharmaceutically acceptable carrier.

(61) A radiopharmaceutical kit comprising a compound of Embodiment 47,or a pharmaceutically acceptable salt form thereof and apharmaceutically acceptable carrier.

(62) A kit of Embodiment 60 further comprising a stabilizer.

(63) A radiopharmaceutical kit according to Embodiment 61, wherein theradioisotope is ¹⁸⁶Re or ¹⁸⁸Re and the kit further comprises one or moreancillary ligands and a reducing agent.

(64) A radiopharmaceutical kit according to Embodiment 63, wherein theancillary ligands are tricine and a phosphine.

(65) A kit according to embodiment 60, further comprising and at leastone agent selected from the group consisting of a chemotherapeutic agentand a radiosensitizer agent, or a pharmaceutically acceptable saltthereof, and a pharmaceutically acceptable carrier.

(66) A kit according to Embodiment 65, wherein the chemotherapeuticagent is selected from the group consisting of mitomycin, tretinoin,ribomustin, gemcitabine, vincristine, etoposide, cladribine,mitobronitol, methotrexate, doxorubicin, carboquone, pentostatin,nitracrine, zinostatin, cetrorelix, letrozole, raltitrexed,daunorubicin, fadrozole, fotemustine, thymalfasin, sobuzoxane,nedaplatin, cytarabine, bicalutamide, vinorelbine, vesnarinone,aminoglutethimide, amsacrine, proglumide, elliptinium acetate,ketanserin, doxifluridine, etretinate, isotretinoin, streptozocin,nimustine, vindesine, flutamide, drogenil, butocin, carmofur, razoxane,sizofilan, carboplatin, mitolactol, tegafur, ifosfamide, prednimustine,picibanil, levamisole, teniposide, improsulfan, enocitabine, lisuride,oxymetholone, tamoxifen, progesterone, mepitiostane, epitiostanol,formestane, interferon-alpha, interferon-2 alpha, interferon-beta,interferon-gamma, colony stimulating factor-1, colony stimulatingfactor-2, denileukin diftitox, interleukin-2, and leutinizing hormonereleasing factor.

(67) A kit according to Embodiment 65, wherein radiosensitizer agent isselected from the group consiting of2-(3-nitro-1,2,4-triazol-1-yl)-N-(2-methoxyethyl)acetamide,N-(3-nitro-4-quinolinyl)-4-morpholinecarboxamidine,3-amino-1,2,4-benzotriazine-1,4-dioxide,N-(2-hydroxyethyl)-2-nitroimidazole-1-acetamide,1-(2-nitroimidazol-1-yl)-3-(1-piperidinyl)-2-propanol, and1-(2-nitro-1-imidazolyl)-3-(1-aziridino)-2-propanol.

(68) A method of treating a pathological disorder mediated by a matrixmetalloproteinase in a patient which comprises administring to a patientin need thereof a therapeutically effective amount of aradiopharmaceutical according to embodiment 47 and a pharmaceuticallyacceptable carrier.

(69) A method of embodiment 68, wherein the disorder is selected fromthe group consisting of atherosclerosis, restenosis, angiogenesis, tumormetastasis, tumor growth, osteoarthritis, and rheumatoid arthritis.

(70) A method of embodiment 68, wherein the disorder is age relatedmacular degeneration, diabetic retinopathy, proliferativevitreoretinopathy,retinopathy of prematurity, ocular tumors, ocularangiogenesis/neovascularization and corneal graft rejection.

(71) A method of embodiment 68, wherein the disorder is cancer selectedfrom the group consisting of prostate, breast, colon, lung melanoma andlymph cancer.

(72) A method of inhibiting proliferation of cancer cells, comprisingcontacting the cancer cells with a proliferation-inhibitory amount of aradiopharmaceutical of embodiment 47.

(73) A method of embodiment 68, wherein the matrix metalloproteinase isselected from the group consiting of: MMP-1, MMP-2, MMP-3, MMP-9, andMMP-14.

(74) A method of embodiment 68 wherein the matrix metalloproteinase isselected from the group consiting of: MMP-2, MMP-9, and MMP-14.

(75) A method of treating cancer in a patient comprising: administeringto a patient in need thereof a therapeutic radiopharmaceutical ofembodiment 47 or a pharmaceutically acceptable salt thereof, and atleast one agent selected from the group consisting of a chemotherapeuticagent and a radiosensitizer agent, or a pharmaceutically acceptable saltthereof.

(76) A method according to embodiment 75 wherein the chemotherapeuticagent is selected from the group consisting of mitomycin, tretinoin,ribomustin, gemcitabine, vincristine, etoposide, cladribine,mitobronitol, methotrexate, doxorubicin, carboquone, pentostatin,nitracrine, zinostatin, cetrorelix, letrozole, raltitrexed,daunorubicin, fadrozole, fotemustine, thymalfasin, sobuzoxane,nedaplatin, cytarabine, bicalutamide, vinorelbine, vesnarinone,aminoglutethimide, amsacrine, proglumide, elliptinium acetate,ketanserin, doxifluridine, etretinate, isotretinoin, streptozocin,nimustine, vindesine, flutamide, drogenil, butocin, carmofur, razoxane,sizofilan, carboplatin, mitolactol, tegafur, ifosfamide, prednimustine,picibanil, levamisole, teniposide, improsulfan, enocitabine, lisuride,oxymetholone, tamoxifen, progesterone, mepitiostane, epitiostanol,formestane, interferon-alpha, interferon-2 alpha, interferon-beta,interferon-gamma, colony stimulating factor-1, colony stimulatingfactor-2, denileukin diftitox, interleukin-2, and leutinizing hormonereleasing factor.

(77) A method according to embodiment 75 wherein the radiosensitizeragent is selected from the group consiting of2-(3-nitro-1,2,4-triazol-1-yl)-N-(2-methoxyethyl)acetamide,N-(3-nitro-4-quinolinyl)-4-morpholinecarboxamidine,3-amino-1,2,4-benzotriazine-1,4-dioxide,N-(2-hydroxyethyl)-2-nitroimidazole-1-acetamide,1-(2-nitroimidazol-1-yl)-3-(1-piperidinyl)-2-propanol, and1-(2-nitro-1-imidazolyl)-3-(1-aziridino)-2-propanol.

(78) A process for the preparation of a radiopharmaceutical, saidprocess comprising generating a macrostructure from a plurality ofmolecular components wherein the plurality of components includes acompound of embodiment 1 and a cytotoxic radioisotope.

(79) A compound as disclosed in any of the examples described herein.

Another aspect of the present invention contemplates the combination ofchemotherapeutics and angiogenesis-targeted therapeuticradiopharmaceuticals of the invention, which target the luminal side ofthe neovasculature of tumors, to provide a surprising, and enhanceddegree of tumor suppression relative to each treatment modality alonewithout significant additive toxicity.

Another aspect of the present invention contemplates the compounds ofthe present invention which is administered in combination therapy, withone or more chemotherapeutic agent(s)selected from the group consistingof mitomycin, tretinoin, ribomustin, gemcitabine, vincristine,etoposide, cladribine, mitobronitol, methotrexate, doxorubicin,carboquone, pentostatin, nitracrine, zinostatin, cetrorelix, letrozole,raltitrexed, daunorubicin, fadrozole, fotemustine, thymalfasin,sobuzoxane, nedaplatin, cytarabine, bicalutamide, vinorelbine,vesnarinone, aminoglutethimide, amsacrine, proglumide, elliptiniumacetate, ketanserin, doxifluridine, etretinate, isotretinoin,streptozocin, nimustine, vindesine, flutamide, drogenil, butocin,carmofur, razoxane, sizofilan, carboplatin, mitolactol, tegafur,ifosfamide, prednimustine, picibanil, levamisole, teniposide,improsulfan, enocitabine, lisuride, oxymetholone, tamoxifen,progesterone, mepitiostane, epitiostanol, formestane, interferon-alpha,interferon-2 alpha, interferon-beta, interferon-gamma, colonystimulating factor-1, colony stimulating factor-2, denileukin diftitox,interleukin-2, and leutinizing hormone releasing factor.

This combination therapy may further, optionally, include aradiosensitizer agent, or a pharmaceutically acceptable salt thereof, toenhance the radiotherapeutic effect together with the chemotherapeuticagent, said radiosensitizer agent being selected from the groupconsisting of2-(3-nitro-1,2,4-triazol-1-yl)-N-(2-methoxyethyl)acetamide,N-(3-nitro-4-quinolinyl)-4-morpholinecarboxamidine,3-amino-1,2,4-benzotriazine-1,4-dioxide,N-(2-hydroxyethyl)-2-nitroimidazole-1-acetamide,1-(2-nitroimidazol-1-yl)-3-(1-piperidinyl)-2-propanol, and1-(2-nitro-1-imidazolyl)-3-(1-aziridino)-2-propanol. A thoroughdiscussion of radiosensitizer agents is provided in the following:Rowinsky-EK, Oncology-Huntingt., 1999 October; 13(10 Suppl 5): 61-70;Chen-AY et al., Oncology-Huntingt. 1999 October; 13(10 Suppl 5): 39-46;Choy-H, Oncology-Huntingt. 1999 October; 13(10 Suppl 5): 23-38; andHerscher-L L et al, Oncology-Huntingt. 1999 October; 13(10 Suppl 5):11-22, which are incorporated herein by reference.

It is a further aspect of the invention to provide kits having aplurality of active ingredients (with or without carrier) which,together, may be effectively utilized for carrying out the novelcombination therapies of the invention.

It is another aspect of the invention to provide a novel pharmaceuticalcomposition which is effective, in and of itself, for utilization in abeneficial combination therapy because it includes compounds of thepresent invention, and a chemotherapeutic agent or a radiosensitizeragent, which may be utilized in accordance with the invention.

In another aspect, the present invention provides a method for treatingcancer in a patient in need of such treatment, said method including thesteps of administering a therapeutically effective amount of a compoundof the present invention and administering a therapeutically effectiveamount of at least one agent selected from the group consisting of achemotherapeutic agent and a radiosensitizer agent.

It is to be understood that this invention covers all appropriatecombinations of the particular and preferred groupings and embodimentsreferred to herein.

DEFINITIONS

The compounds herein described may have asymmetric centers. Unlessotherwise indicated, all chiral, diastereomeric and racemic forms areincluded in the present invention. Many geometric isomers of olefins,C═N double bonds, and the like can also be present in the compoundsdescribed herein, and all such stable isomers are contemplated in thepresent invention. It will be appreciated that compounds of the presentinvention contain asymmetrically substituted carbon atoms, and may beisolated in optically active or racemic forms. It is well known in theart how to prepare optically active forms, such as by resolution ofracemic forms or by synthesis from optically active starting materials.Two distinct isomers (cis and trans) of the peptide bond are known tooccur; both can also be present in the compounds described herein, andall such stable isomers are contemplated in the present invention. The Dand L-isomers of a particular amino acid are designated herein using theconventional 3-letter abbreviation of the amino acid, as indicated bythe following examples: D-Leu, or L-Leu.

When any variable occurs more than one time in any substituent or in anyformula, its definition on each occurrence is independent of itsdefinition at every other occurrence. Thus, for example, if a group isshown to be substituted with 0-2 R⁵², then said group may optionally besubstituted with up to two R⁵², and R⁵² at each occurrence is selectedindependently from the defined list of possible R⁵². Also, by way ofexample, for the group —N(R⁵³)₂, each of the two R⁵³ substituents on Nis independently selected from the defined list of possible R⁵³.Combinations of substituents and/or variables are permissible only ifsuch combinations result in stable compounds. When a bond to asubstituent is shown to cross the bond connecting two atoms in a ring,then such substituent may be bonded to any atom on the ring.

The term “metallopharmaceutical” means a pharmaceutical comprising ametal. The metal is the cause of the imageable signal in diagnosticapplications and the source of the cytotoxic radiation inradiotherapeutic applications. Radiopharmaceuticals aremetallopharmaceuticals in which the metal is a radioisotope.

By “reagent” is meant a compound of this invention capable of directtransformation into a metallopharmaceutical of this invention. Reagentsmay be utilized directly for the preparation of themetallopharmaceuticals of this invention or may be a component in a kitof this invention.

The term “binding agent” means a metallopharmaceutical of this inventionhaving affinity for and capable of binding to a matrixmetalloproteinase. The binding agents of this invention have Ki<1000 nm,more preferabley Ki<100 nM.

By “stable compounds or “stable structure” is meant herein a compoundthat is sufficiently robust to survive isolation to a useful degree ofpurity from a reaction mixture, and formulation into an efficaciouspharmaceutical agent.

The term “substituted”, as used herein, means that one or more hydrogenson the designated atom or group is replaced with a selection from theindicated group, provided that the designated atom's or group's normalvalency is not exceeded, and that the substitution results in a stablecompound. When a substituent is keto (i.e., ═O), then 2 hydrogens on theatom are replaced.

The term “bond”, as used herein, means either a single or double bond.

The term “salt”, as used herein, is used as defined in the CRC Handbookof Chemistry and Physics, 65th Edition, CRC Press, Boca Raton, Fla.,1984, as any substance which yields ions, other than hydrogen orhydroxyl ions. As used herein, “pharmaceutically acceptable salts” referto derivatives of the disclosed compounds modified by making acid orbase salts. Examples of pharmaceutically acceptable salts include, butare not limited to, mineral or organic acid salts of basic residues suchas amines; alkali or organic salts of acidic residues such as carboxylicacids; and the like.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable prodrugs” as used herein meansthose prodrugs of the compounds useful according to the presentinvention which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswith undue toxicity, irritation, allergic response, and the like,commensurate with a reasonable benefit/risk ratio, and effective fortheir intended use, as well as the zwitterionic forms, where possible,of the compounds of the invention. The term “prodrug” means compoundsthat are rapidly transformed in vivo to yield the parent compound of theabove formula, for example by hydrolysis in blood. Functional groupswhich may be rapidly transformed, by metabolic cleavage, in vivo form aclass of groups reactive with the carboxyl group of the compounds ofthis invention. They include, but are not limited to such groups asalkanoyl (such as acetyl, propionyl, butyryl, and the like),unsubstituted and substituted aroyl (such as benzoyl and substitutedbenzoyl), alkoxycarbonyl (such as ethoxycarbonyl), trialkylsilyl (suchas trimethyl- and triethysilyl), monoesters formed with dicarboxylicacids (such as succinyl), and the like. Because of the ease with whichthe metabolically cleavable groups of the compounds useful according tothis invention are cleaved in vivo, the compounds bearing such groupsact as pro-drugs. The compounds bearing the metabolically cleavablegroups have the advantage that they may exhibit improved bioavailabilityas a result of enhanced solubility and/or rate of absorption conferredupon the parent compound by virtue of the presence of the metabolicallycleavable group. A thorough discussion of prodrugs is provided in thefollowing: Design of Prodrugs, H. Bundgaard, ed., Elsevier, 1985;Methods in Enzymology, K. Widder et al, Ed., Academic Press, 42,p.309-396, 1985; A Textbook of Drug Design and Development,Krogsgaard-Larsen and H. Bundgaard, ed., Chapter 5; “Design andApplications of Prodrugs” p.113-191, 1991; Advanced Drug DeliveryReviews, H. Bundgard, 8, p.1-38, 1992; Journal of PharmaceuticalSciences, 77, p. 285, 1988; Chem. Pharm. Bull., N. Nakeya et al, 32, p.692, 1984; Pro-drugs as Novel Delivery Systems, T. Higuchi and V.Stella, Vol. 14 of the A.C.S. Symposium Series, and BioreversibleCarriers in Drug Design, Edward B. Roche, ed., American PharmaceuticalAssociation and Pergamon Press, 1987, which are incorporated herein byreference.

As used herein, “pharmaceutically acceptable salts” refer to derivativesof the disclosed compounds wherein the parent compound is modified bymaking acid or base salts thereof. Examples of pharmaceuticallyacceptable salts include, but are not limited to, mineral or organicacid salts of basic residues such as amines; alkali or organic salts ofacidic residues such as carboxylic acids; and the like. Thepharmaceutically acceptable salts include the conventional non-toxicsalts or the quaternary ammonium salts of the parent compound formed,for example, from non-toxic inorganic or organic acids. For example,such conventional non-toxic salts include those derived from inorganicacids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric,nitric and the like; and the salts prepared from organic acids such asacetic, propionic, succinic, glycolic, stearic, lactic, tartaric,citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic,benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric,toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic,and the like.

The pharmaceutically acceptable salts of the present invention can besynthesized from the parent compound which contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; generally, nonaqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrileare preferred. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,1985, p. 1418, the disclosure of which is hereby incorporated byreference.

As used herein, “alkyl” is intended to include both branched andstraight-chain saturated aliphatic hydrocarbon groups having thespecified number of carbon atoms, examples of which include, but are notlimited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl,sec-butyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, and decyl;“cycloalkyl” or “carbocycle” is intended to include saturated andpartially unsaturated ring groups, including mono-, bi- or poly-cyclicring systems, such as-cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclooctyl and adamantyl; “bicycloalkyl” or “bicyclic” isintended to include saturated bicyclic ring groups such as[3.3.0]bicyclooctane, [4.3.0]bicyclononane, [4.4.0]bicyclodecane(decalin), [2.2.2]bicyclooctane, and so forth.

As used herein, the term “alkene” or “alkenyl” is intended to includehydrocarbon chains having the specified number of carbon atoms of eithera straight or branched configuration and one or more unsaturatedcarbon-carbon bonds which may occur in any stable point along the chain,such as ethenyl, propenyl, and the like.

As used herein, the term “alkyne” or “alkynyl” is intended to includehydrocarbon chains having the specified number of carbon atoms of eithera straight or branched configuration and one or more unsaturatedcarbon-carbon triple bonds which may occur in any stable point along thechain, such as propargyl, and the like.

As used herein, “aryl” or “aromatic residue” is intended to mean phenylor naphthyl, which when substituted, the substitution can be at anyposition.

As used herein, the term “heterocycle” or “heterocyclic system” isintended to mean a stable 5- to 7-membered monocyclic or bicyclic or 7-to 10-membered bicyclic heterocyclic ring which is saturated partiallyunsaturated or unsaturated (aromatic), and which consists of carbonatoms and from 1 to 4 heteroatoms independently selected from the groupconsisting of N, O and S and including any bicyclic group in which anyof the above-defined heterocyclic rings is fused to a benzene ring. Thenitrogen and sulfur heteroatoms may optionally be oxidized. Theheterocyclic ring may be attached to its pendant group at any heteroatomor carbon atom which results in a stable structure. The heterocyclicrings described herein may be substituted on carbon or on a nitrogenatom if the resulting compound is stable. If specifically noted, anitrogen in the heterocycle may optionally be quaternized. It ispreferred that when the total number of S and O atoms in the heterocycleexceeds 1, then these heteroatoms are not adjacent to one another. It ispreferred that the total number of S and O atoms in the heterocycle isnot more than 1. As used herein, the term “aromatic heterocyclic system”is intended to mean a stable 5- to 7-membered monocyclic or bicyclic or7- to 10-membered bicyclic heterocyclic aromatic ring which consists ofcarbon atoms and from 1 to 4 heteroatoms independently selected from thegroup consisting of N, O and S. It is preferred that the total number ofS and O atoms in the aromatic heterocycle is not more than 1.

Examples of heterocycles include, but are not limited to, 1H-indazole,2-pyrrolidonyl, 2H,6H-1,5,2-dithiazinyl, 2H-pyrrolyl, 3H-indolyl,4-piperidonyl, 4aH-carbazole, 4H-quinolizinyl, 6H-1,2,5-thiadiazinyl,acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl,benzothiophenyl, benzoxazolyl, benzthiazolyl, benztriazolyl,benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazalonyl,carbazolyl, 4aH-carbazolyl, β-carbolinyl, chromanyl, chromenyl,cinnolinyl, decahydroquinolinyl, 2H, 6H-1,5,2-dithiazinyl,dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl,imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl,indolizinyl, indolyl, isobenzofuranyl, isochromanyl, isoindazolyl,isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl,morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl,1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl,1,3,4-oxadiazolyl, oxazolidinyl., oxazolyl, oxazolidinylperimidinyl,phenanthridinyl, phenanthrolinyl, phenarsazinyl, phenazinyl,phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, piperazinyl,piperidinyl, pteridinyl, piperidonyl, 4-piperidonyl, pteridinyl,purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl,pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl,pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, pyrrolyl, quinazolinyl,quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, carbolinyl,tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl,6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl,1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl,thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl,triazinyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,5-triazolyl,1,3,4-triazolyl, xanthenyl. Preferred heterocycles include, but are notlimited to, pyridinyl, furanyl, thienyl, pyrrolyl, pyrazolyl,imidazolyl, indolyl, benzimidazolyl, 1H-indazolyl, oxazolidinyl,benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl, or isatinoyl.Also included are fused ring and spiro compounds containing, forexample, the above heterocycles.

As used herein, the term “alkaryl” means an aryl group bearing an alkylgroup of 1-10 carbon atoms; the term “aralkyl” means an alkyl group of1-10 carbon atoms bearing an aryl group; the term “arylalkaryl” means anaryl group bearing an alkyl group of 1-10 carbon atoms bearing an arylgroup; and the term “heterocycloalkyl” means an alkyl group of 1-10carbon atoms bearing a heterocycle.

A “polyalkylene glycol” is a polyethylene glycol, polypropylene glycolor polybutylene glycol having a molecular weight of less than about5000, terminating in either a hydroxy or alkyl ether moiety.

A “carbohydrate” is a polyhydroxy aldehyde, ketone, alcohol or acid, orderivatives thereof, including polymers thereof having polymericlinkages of the acetal type.

A “cyclodextrin” is a cyclic oligosaccharide. Examples of cyclodextrinsinclude, but are not limited to, α-cyclodextrin,hydroxyethyl-α-cyclodextrin, hydroxypropyl-α-cyclodextrin,β-cyclodextrin, hydroxypropyl-α-cyclodextrin,carboxymethyl-β-cyclodextrin, dihydroxypropyl-β-cyclodextrin,hydroxyethyl-β-cyclodextrin, 2,6 di-O-methyl-α-cyclodextrin,sulfated-β-cyclodextrin, γ-cyclodextrin, hydroxypropyl-γ-cyclodextrin,dihydroxypropyl-γ-cyclodextrin, hydroxyethyl-γ-cyclodextrin, andsulfated γ-cyclodextrin.

As used herein, the term “polycarboxyalkyl” means an alkyl group havingbetween two and about 100 carbon atoms and a plurality of carboxylsubstituents; and the term “polyazaalkyl” means a linear or branchedalkyl group having between two and about 100 carbon atoms, interruptedby or substituted with a plurality of amine groups.

A “reducing agent” is a compound that reacts with a radionuclide, whichis typically obtained as a relatively unreactive, high oxidation statecompound, to lower its oxidation state by transferring electron(s) tothe radionuclide, thereby making it more reactive. Reducing agentsuseful in the preparation of radiopharmaceuticals and in diagnostic kitsuseful for the preparation of said radiopharmaceuticals include but arenot limited to stannous chloride, stannous fluoride, formamidinesulfinic acid, ascorbic acid, cysteine, phosphines, and cuprous orferrous salts. Other reducing agents are described in Brodack et. al.,PCT Application 94/22496, which is incorporated herein by reference.

A “transfer ligand” is a ligand that forms an intermediate complex witha metal ion that is stable enough to prevent unwanted side-reactions butlabile enough to be converted to a metallopharmaceutical. The formationof the intermediate complex is kinetically favored while the formationof the metallopharmaceutical is thermodynamically favored. Transferligands useful in the preparation of metallopharmaceuticals and indiagnostic kits useful for the preparation of diagnosticradiopharmaceuticals include but are not limited to gluconate,glucoheptonate, mannitol, glucarate,N,N,N′,N′-ethylenediaminetetraacetic acid, pyrophosphate andmethylenediphosphonate. In general, transfer ligands are comprised ofoxygen or nitrogen donor atoms.

The term “donor atom” refers to the atom directly attached to a metal bya chemical bond. “Ancillary” or “co-ligands” are ligands that areincorporated into a radiopharmaceutical during its synthesis. They serveto complete the coordination sphere of the radionuclide together withthe chelator or radionuclide bonding unit of the reagent. Forradiopharmaceuticals comprised of a binary ligand system, theradionuclide coordination sphere is composed of one or more chelators orbonding units from one or more reagents and one or more ancillary orco-ligands, provided that there are a total of two types of ligands,chelators or bonding units. For example, a radiopharmaceutical comprisedof one chelator or bonding unit from one reagent and two of the sameancillary or co-ligands and a radiopharmaceutical comprised of twochelators or bonding units from one or two reagents and one ancillary orco-ligand are both considered to be comprised of binary ligand systems.For radiopharmaceuticals comprised of a ternary ligand system, theradionuclide coordination sphere is composed of one or more chelators orbonding units from one or more reagents and one or more of two differenttypes of ancillary or co-ligands, provided that there are a total ofthree types of ligands, chelators or bonding units. For example, aradiopharmaceutical comprised of one chelator or bonding unit from onereagent and two different ancillary or co-ligands is considered to becomprised of a ternary ligand system.

Ancillary or co-ligands useful in the preparation ofradiopharmaceuticals and in diagnostic kits useful for the preparationof said radiopharmaceuticals are comprised of one or more oxygen,nitrogen, carbon, sulfur, phosphorus, arsenic, selenium, and telluriumdonor atoms. A ligand can be a transfer ligand in the synthesis of aradiopharmaceutical and also serve as an ancillary or co-ligand inanother radiopharmaceutical. Whether a ligand is termed a transfer orancillary or co-ligand depends on whether the ligand remains in theradionuclide coordination sphere in the radiopharmaceutical, which isdetermined by the coordination chemistry of the radionuclide and thechelator or bonding unit of the reagent or reagents.

A “chelator” or “bonding unit” is the moiety or group on a reagent thatbinds to a metal ion through the formation of chemical bonds with one ormore donor atoms.

The term “binding site” means the site in vivo or in vitro that binds abiologically active molecule.

A “diagnostic kit” or “kit” comprises a collection of components, termedthe formulation, in one or more vials which are used by the practicingend user in a clinical or pharmacy setting to synthesize diagnosticradiopharmaceuticals. The kit provides all the requisite components tosynthesize and use the diagnostic radiopharmaceutical except those thatare commonly available to the practicing end user, such as water orsaline for injection, a solution of the radionuclide, equipment forheating the kit during the synthesis of the radiopharmaceutical, ifrequired, equipment necessary for administering the radiopharmaceuticalto the patient such as syringes and shielding, and imaging equipment.

Therapeutic radiopharmaceuticals, X-ray contrast agent pharmaceuticals,ultrasound contrast agent pharmaceuticals and metallopharmaceuticals formagnetic resonance imaging contrast are provided to the end user intheir final form in a formulation contained typically in one vial, aseither a lyophilized solid or an aqueous solution. The end userreconstitutes the lyophilized with water or saline and withdraws thepatient dose or just withdraws the dose from the aqueous solutionformulation as provided.

A “lyophilization aid” is a component that has favorable physicalproperties for lyophilization, such as the glass transition temperature,and is added to the formulation to improve the physical properties ofthe combination of all the components of the formulation forlyophilization.

A “stabilization aid” is a component that is added to themetallopharmaceutical or to the diagnostic kit either to stabilize themetallopharmaceutical or to prolong the shelf-life of the kit before itmust be used. Stabilization aids can be antioxidants, reducing agents orradical scavengers and can provide improved stability by reactingpreferentially with species that degrade other components or themetallopharmaceutical.

A “solubilization aid” is a component that improves the solubility ofone or more other components in the medium required for the formulation.

A “bacteriostat” is a component that inhibits the growth of bacteria ina formulation either during its storage before use of after a diagnostickit is used to synthesize a radiopharmaceutical.

The following abbreviations are used herein:

Acm acetamidomethyl b-Ala, beta-Ala 3-aminopropionic acid or bAla ATA2-aminothiazole-5-acetic acid or 2- aminothiazole-5-acetyl group Boct-butyloxycarbonyl CBZ, Cbz or Z Carbobenzyloxy Cit citrulline Dap2,3-diaminopropionic acid DCC dicyclohexylcarbodiimide DIEAdiisopropylethylamine DMAP 4-dimethylaminopyridine EOE ethoxyethyl HBTU2-(1H-Benzotriazol-1-yl)-1,1,3,3- tetramethyluronium hexafluorophosphatehynic boc-hydrazinonicotinyl group or 2-[[[5-[carbonyl]-2-pyridinyl]hydrazono]methyl]- benzenesulfonic acid, NMeArgor MeArg a-N-methyl arginine NMeAsp a-N-methyl aspartic acid NMMN-methylmorpholine OcHex O-cyclohexyl OBzl O-benzyl oSu O-succinimidylTBTU 2-(1H-Benzotriazol-1-yl)-1,1,3,3- tetramethyluroniumtetrafluoroborate THF tetrahydrofuranyl THP tetrahydropyranyl Tos tosylTr trityl

The following conventional three-letter amino acid abbreviations areused herein; the conventional one-letter amino acid abbreviations areNOT used herein:

-   -   Ala=alanine    -   Arg=arginine    -   Asn=asparagine    -   Asp=aspartic acid    -   Cys=cysteine    -   Gln=glutamine

-   Glu=glutamic acid

-   Gly=glycine    -   Mis=histidine    -   Ile=isoleucine    -   Leu=leucine    -   Lys=lysine    -   Met=methionine    -   Nle=norleucine    -   Orn=ornithine    -   Phe=phenylalanine    -   Phg=phenylglycine    -   Pro=proline    -   Sar=sarcosine    -   Ser=serine    -   Thr=threonine    -   Trp=tryptophan    -   Tyr=tyrosine    -   Val=valine

Matrix metalloproteinase (MMP) activity and extracellular matrixdegradation is dependent on the comparative balance between MMPs andTIMPs. Elevated TIMP activity suppresses angiogenesis via inhibition ofendothelial cell migration. TIMPs and synthetic small molecules ormatrix metalloproteinase inhibitors have therapeutic potential fordiseases involving elevated levels of MMP activity (Whittaker, M. et al,Chem. Rev., 1999, 99, 2735-2776; Babine, R. E. et al, Chem. Rev., 1997,97, 1359; De, B. et al, Ann. N.Y. Acad. Sci., 1999, 878, 40-60; Summers,J. B. et al, Annual Reports in Med. Chem., 1998, 33, 131).

A functional group, such as —CONH—OH, —COOH, or —SH, is necessary for amolecule to be an effective inhibitor of MMPs. This functional group isinvolved in the chelation of the active site zinc ion, and is commonlyreferred to as the zinc binding group or ZBG. The hydroxamate, forexample, is a bidentate ligand for zinc.

One of the most studied classes of MMPIs is the succinyl hydroxamates. Ageneric structure of succinyl hydroxamates is shown below (1).

The ethylene spacer between the ZBG (—CONH—OH) and the succinyl amide isessential for potent activity. Substitution at P₁ tends to conferbroad-spectrum activity on the MMPIs. Substituents at this position, ingeneral, tend to point away from the enzyme. Moieties capable ofhydrogen bonding and lipophilic substituents at the P₁ position a to thehydroxamate (Johnson, W. H. et al, J. Enz. Inhib., 1987, 2, 1) tend toenhance activity (2). Incorporation of a hydroxyl group (Beckett, P. R.,et al, Drug Discovery Today, 1996, 1, 16) at that position improves oralactivity in some case (3).

Substituents at the P₁′ position on the succinyl hydroxamates tend toimpart selectivity to the MMPIs. The S₁′ pocket is deep for MMP-2,MMP-3, MMP-8 and MMP-9 and occluded (short) for MMP-1 and MMP-7. A longalkyl substituent at the P₁′ position, for example, imparts selectivity(Miller, A. et al, Bioorg. Med. Chem. Lett., 1997, 7, 193) for MMP-2over MMP-1 and MMP-3 (4 and 5).

Substituents at the P₂′ position also point away from the enzyme. The P₁and the P₂′ positions can be linked (Xue, C-B. et al, J. Med. Chem.,1998, 41, 1745; Steinman, D. H. et al, Bioorg. Med. Chem. Lett., 1998,8, 2087) to form a macrocycle (6). Compounds such as (6) also exhibitnanomolar activity.

The nature of the macrocycle can impart some selectivity against MMPIs.The P₂′ and the P₃′ positions may be cyclized to form lactams. The sizeof the lactam governs the selectivity.

Other succinyl hydroxamates with modified P₂′ and P₃′ positions, such as(8) also have shown potent inhibition of MMP's. Those compounds andsyntheses of them are further described in the following patentapplications which are hereby incorporated by reference into this patentapplication: U.S. patent application Ser. No. 08/743,439 and U.S. Pat.Nos. 6,057,336, 6,576,664, 6,455,522, 6,429,213, 6,365,587, 6,268,379,6,495,548, and 6,376,665.

Another class of MMPIs is the sulfonamide hydroxamates, such as (9) and(10). Modification of the isopropyl substituent in (10) results in deeppocket MMP selectivity, for example MMP-2 vs MMP-1 (Santos, O. et al.,J. Clin. Exp. Metastasis, 1997, 15, 499; MacPherson, L. J. et al, J.Med. Chem., 1997, 40, 2525).

Selectivity for MMP-2 and MMP-9 was observed in the derivatized‘alanine’ hydroxamates, such as compounds (11) and (12). The P₁ positionis available for limited modification as described in the patents andapplications incorporated by reference above.

Other compounds with selectivity for MMP-2 and MMP-9 over MMP-1 include(13). In this example the alpha position has a quaternary carbon and themolecule does not contain any stereo centers (Lovejoy, B. et al., NatureStruct. Biol., 1999, 6, 217).

In the non-hydroxamate series a number of compounds have been reportedwith a variety of structures. Use of carboxylic acid as the ZBG has alsoreceived attention. In the case of compound (14), significantselectivity for MMP-2 (vs MMP-1) was observed when X=butyl vs X=H(Sahoo, S. P. et al, Bioorg. Med. Chem. Lett., 1995, 5, 2441).

Although thiols are monodentate ZBGs, some succinyl thiols such as (15)have exhibited good activity (Levin, J. I. et al, Bioorg. Med. Chem.Lett., 1998, 8, 1163). The P₃′ position may be utilized to conjugate avariety of linkers and chelators (as described above) for thepreparation of diagnostic agents.

The pharmaceuticals of the present invention, containing a liganddirected at one or more MMP's (e.g. MMP-1, MMP-2, MMP-3, MMP-9), willlocalize a diagnostic imaging probe or cytotoxic radioisotope to thesite of pathology for the purpose of non-invasive imaging or treatmentof these diseases. The imaging agent may be a MMP inhibitor/antagonistlinked to radioisotopes which are known to be useful for imaging bygamma scintigraphy (Tc-99m, In-111, I-123 and others). Alternatively,the MMP targeting ligand could be bound to a single or multivalentchelator moiety for attachment of one or more Gadolinium, Manganese, orother paramagnetic metal atoms, which would cause a local change inmagnetic properties, such as relaxivity or susceptibility, at the siteof tissue damage, which could then be imaged with magnetic resonanceimaging systems. Alternatively, the MMP inhibitor/antagonist would bebound to a phospholipid or polymer material which would be used toencapsulate/stabilize microspheres of gas which would be detectable byultrasound imaging following localization at the site of tissue injury.

Therapeutic radiopharmaceuticals of the present invention comprised of aMMP inhibitor labeled with a radioisotope that emits a beta particle, analpha particle or Auger electrons, localize in tumors selectively anddeliver a cytotoxic dose of radiation to the tumor to treat the disease.

Multiple therapy comprises the use of the therapeuticradiopharmaceuticals of the present invention in combination with thecompounds from the lists below which include chemotherapeutics,immunomodulators or colony-stimulating factors. The chemotherapeuticdrugs include: Cytotoxics: mitomycin, tretinoin, ribomustin,gemcitabine, vincristine, etoposide, cladribine, mitobronitol,methotrexate, doxorubicin, carboquone, pentostatin, nitracrine,zinostatin, cetrorelix, letrozole, raltitrexed, daunorubicin, fadrozole,fotemustine, thymalfasin, sobuzoxane, nedaplatin, cytarabine,bicalutamide, vinorelbine, vesnarinone, aminoglutethimide, amsacrine,proglumide, elliptinium acetate, ketanserin, doxifluridine, etretinate,isotretinoin, streptozocin, nimustine, vindesine, flutamide, drogenil,butocin, carmofur, razoxane, sizofilan, carboplatin, mitolactol,tegafur, ifosfamide, prednimustine, picibanil, levamisole, teniposide,improsulfan, enocitabine, and lisuride. The steroids include:oxymetholone, tamoxifen, progesterone, mepitiostane, epitiostanol,formestane. The biologics include: interferon-alpha, interferon-2 alpha,interferon-beta, interferon-gamma, colony stimulating factor-1, colonystimulating factor-2, denileukin diftitox, interleukin-2, leutinizinghormone releasing factor.

The ultrasound contrast agents of the present invention comprise aplurality of matrix metalloproteinase inhibiting moieties attached to orincorporated into a microbubble of a biocompatible gas, a liquidcarrier, and a surfactant microsphere, further comprising an optionallinking moiety, L_(n), between the targeting moieties and themicrobubble. In this context, the term liquid carrier means aqueoussolution and the term surfactant means any amphiphilic material whichproduces a reduction in interfacial tension in a solution. A list ofsuitable surfactants for forming surfactant microspheres is disclosed inEP0727225A2, herein incorporated by reference. The term surfactantmicrosphere includes nanospheres, liposomes, vesicles and the like. Thebiocompatible gas can be air, or a fluorocarbon, such as a C₃-C₅perfluoroalkane, which provides the difference in echogenicity and thusthe contrast in ultrasound imaging. The gas is encapsulated or containedin the microsphere to which is attached the biodirecting group,optionally via a linking group. The attachment can be covalent, ionic orby van der Waals forces. Specific examples of such contrast agentsinclude lipid encapsulated perfluorocarbons with a plurality of tumorneovasculature receptor binding peptides, polypeptides orpeptidomimetics.

X-ray contrast agents of the present invention are comprised of one ormore matrix metalloproteinase inhibiting targeting moieties attached toone or more X-ray absorbing or “heavy” atoms of atomic number 20 orgreater, further comprising an optional linking moiety, L_(n), betweenthe targeting moieties and the X-ray absorbing atoms. The frequentlyused heavy atom in X-ray contrast agents is iodine. Recently, X-raycontrast agents comprised of metal chelates (Wallace, R., U.S. Pat. No.5,417,959) and polychelates comprised of a plurality of metal ions(Love, D., U.S. Pat. No. 5,679,810) have been disclosed. More recently,multinuclear cluster complexes have been disclosed as X-ray contrastagents (U.S. Pat. No. 5,804,161, PCT WO91/14460, and PCT WO 92/17215).

MRI contrast agents of the present invention are comprised of one ormore matrix metalloproteinase inhibiting targeting moieties attached toone or more paramagnetic metal ions, further comprising an optionallinking moiety, L_(n), between the targeting moieties and theparamagnetic metal ions. The paramagnetic metal ions are present in theform of metal complexes or metal oxide particles. U.S. Pat. Nos.5,412,148, and 5,760,191, describe examples of chelators forparamagnetic metal ions for use in MRI contrast agents. U.S. Pat. No.5,801,228, U.S. Pat. No. 5,567,411, and U.S. Pat. No. 5,281,704,describe examples of polychelants useful for complexing more than oneparamagnetic metal ion for use in MRI contrast agents. U.S. Pat. No.5,520,904, describes particulate compositions comprised of paramagneticmetal ions for use as MRI contrast agents.

Administration of a compound of the present invention in combinationwith such additional therapeutic agents, may afford an efficacyadvantage over the compounds and agents alone, and may do so whilepermitting the use of lower doses of each. A lower dosage minimizes thepotential of side effects, thereby providing an increased margin ofsafety. The combination of a compound of the present invention with suchadditional therapeutic agents is preferably a synergistic combination.Synergy, as described for example by Chou and Talalay, Adv. EnzymeRegul. 22: 27-55 (1984), occurs when the therapeutic effect of thecompound and agent when administered in combination is greater than theadditive effect of the either the compound or agent when administeredalone. In general, a synergistic effect is most clearly demonstrated atlevels that are (therapeutically) sub-optimal for either the compound ofthe present invention, a chemotherapeutic agent or a radiosensitizeragent alone, but which are highly efficacious in combination. Synergycan be in terms of improved tumor response without substantial increasesin toxicity over individual treatments alone, or some other beneficialeffect of the combination compared with the individual components.

The compounds of the present invention, and a chemotherapeutic agent ora radiosensitizer agent, utilized in combination therapy may beadministered simultaneously, in either separate or combinedformulations, or at different times e.g., sequentially, such that acombined effect is achieved. The amounts and regime of administrationwill be adjusted by the practitioner, by preferably initially loweringtheir standard doses and then titrating the results obtained.

The invention also provides kits or single packages combining two ormore active ingredients useful in treating cancer. A kit may provide(alone or in combination with a pharmaceutically acceptable diluent orcarrier), the compound of the present invention and additionally atleast one agent selected from the group consisting of a chemotherapeuticagent and a radiosensitizer agent (alone or in combination with diluentor carrier).

The pharmaceuticals of the present invention have the formulae,(Q)_(d)—L_(n)—(C_(h)—X), (Q)_(d)—L_(n)—(C_(h)—X¹)_(d′),(Q)_(d)—L_(n)—(X²)_(d″), and (Q)_(d)—L_(n)—(X³), wherein Q represents apeptide, polypeptide, peptidomimetic or non-peptide that binds to amatrix metalloproteinase, d is 1-10, L_(n) represents an optionallinking group, C_(h) represents a metal chelator or bonding moiety, Xrepresents a radioisotope, X¹ represents paramagnetic metal ion, X²represents a paramagnetic metal ion or heavy atom containing insolublesolid particle, d″ is 1-100, and X³ represents a surfactant microsphereof an echogenic gas.

Preferred pharmaceuticals of the present invention are comprised ofinhibitors, Q, which exhibit selectivity for MMP-1, MMP-2, MMP-3, MMP-9,or MMP-14 alone or in combination over the other MMPs. Examples ofpreferred moieties, Q, include compounds 4, 5, 6, 8, 9, 10, 11, 12, and13.

Most preferred are comprised of inhibitors, Q, which exhibit selectivityfor MMP-2, MMP-9, or MMP-14 alone or in combination over the other MMPs.Examples of the most preferred moieties, Q, include compounds 6, 8, 11,and 12.

The pharmaceuticals of the present invention can be synthesized byseveral approaches. One approach involves the synthesis of the targetingpeptide, polypeptide, peptidomimetic, or non-peptide moiety, Q, anddirect attachment of one or more moieties, Q, to one or more metalchelators or bonding moieties, C_(h), or to a paramagnetic metal ion orheavy atom containing solid particle, or to an echogenic gasmicrobubble. Another approach involves the attachment of one or moremoieties, Q, to the linking group, L_(n), which is then attached to oneor more metal chelators or bonding moieties, C_(h), or to a paramagneticmetal ion or heavy atom containing solid particle, or to an echogenicgas microbubble. Another approach, useful in the synthesis ofpharmaceuticals wherein d is 1, involves the synthesis of the moiety,Q—L_(n), together, by incorporating an amino acid or amino acid mimeticresidue bearing L_(n) into the synthesis of the peptide, polypeptide,peptidomimetic, or non-peptide. The resulting moiety, Q—L_(n), is thenattached to one or more metal chelators or bonding moieties, C_(h), orto a paramagnetic metal ion or heavy atom containing solid particle, orto an echogenic gas microbubble. Another approach involves the synthesisof a peptide, polypeptide, peptidomimetic, or non-peptide, Q, bearing afragment of the linking group, L_(n), one or more of which are thenattached to the remainder of the linking group and then to one or moremetal chelators or bonding moieties, C_(h), or to a paramagnetic metalion or heavy atom containing solid particle, or to an echogenic gasmicrobubble.

The peptides, polypeptides, peptidomimetics, or non-peptide, Q,optionally bearing a linking group, L_(n), or a fragment of the linkinggroup, can be synthesized using standard synthetic methods known tothose skilled in the art. Preferred methods include but are not limitedto those methods described below.

Generally, peptides, polypeptides and peptidomimetics are elongated bydeprotecting the alpha-amine of the C-terminal residue and coupling thenext suitably protected amino acid through a peptide linkage using themethods described. This deprotection and coupling procedure is repeateduntil the desired sequence is obtained. This coupling can be performedwith the constituent amino acids in a stepwise fashion, or condensationof fragments (two to several amino acids), or combination of bothprocesses, or by solid phase peptide synthesis according to the methodoriginally described by Merrifield, J. Am. Chem. Soc., 85, 2149-2154(1963), the disclosure of which is hereby incorporated by reference.

The peptides, polypeptides and peptidomimetics may also be synthesizedusing automated synthesizing equipment. In addition to the foregoing,procedures for peptide, polypeptide and peptidomimetic synthesis aredescribed in Stewart and Young, “Solid Phase Peptide Synthesis”, 2nd ed,Pierce Chemical Co., Rockford, Ill. (1984); Gross, Meienhofer,Udenfriend, Eds., “The Peptides: Analysis, Synthesis, Biology, Vol. 1,2, 3, 5, and 9, Academic Press, New York, (1980-1987); Bodanszky,“Peptide Chemistry: A Practical Textbook”, Springer-Verlag, New York(1988); and Bodanszky et al. “The Practice of Peptide Synthesis”Springer-Verlag, New York (1984), the disclosures of which are herebyincorporated by reference.

The coupling between two amino acid derivatives, an amino acid and apeptide, polypeptide or peptidomimetic, two peptide, polypeptide orpeptidomimetic fragments, or the cyclization of a peptide, polypeptideor peptidomimetic can be carried out using standard coupling proceduressuch as the azide method, mixed carbonic acid anhydride (isobutylchloroformate) method, carbodiimide (dicyclohexylcarbodiimide,diisopropylcarbodiimide, or water-soluble carbodiimides) method, activeester (p-nitrophenyl ester, N-hydroxysuccinic imido ester) method,Woodward reagent K method, carbonyldiimidazole method, phosphorusreagents such as BOP-Cl, or oxidation-reduction method. Some of thesemethods (especially the carbodiimide) can be enhanced by the addition of1-hydroxybenzotriazole. These coupling reactions may be performed ineither solution (liquid phase) or solid phase.

The functional groups of the constituent amino acids or amino acidmimetics must be protected during the coupling reactions to avoidundesired bonds being formed. The protecting groups that can be used arelisted in Greene, “Protective Groups in Organic Synthesis” John Wiley &Sons, New York (1981) and “The Peptides: Analysis, Synthesis, Biology,Vol. 3, Academic Press, New York (1981), the disclosure of which ishereby incorporated by reference. The alpha-carboxyl group of theC-terminal residue is usually protected by an ester that can be cleavedto give the carboxylic acid. These protecting groups include: 1) alkylesters such as methyl and t-butyl, 2) aryl esters such as benzyl andsubstituted benzyl, or 3) esters which can be cleaved by mild basetreatment or mild reductive means such as trichloroethyl and phenacylesters. In the solid phase case, the C-terminal amino acid is attachedto an insoluble carrier (usually polystyrene). These insoluble carrierscontain a group which will react with the carboxyl group to form a bondwhich is stable to the elongation conditions but readily cleaved later.Examples of which are: oxime resin (DeGrado and Kaiser (1980) J. Org.Chem. 45, 1295-1300) chloro or bromomethyl resin, hydroxymethyl resin,and aminomethyl resin. Many of these resins are commercially availablewith the desired C-terminal amino acid already incorporated.

The alpha-amino group of each amino acid must be protected. Anyprotecting group known in the art can be used. Examples of these are: 1)acyl types such as formyl, trifluoroacetyl, phthalyl, andp-toluenesulfonyl; 2) aromatic carbamate types such as benzyloxycarbonyl(Cbz) and substituted benzyloxycarbonyls,1-(p-biphenyl)-1-methylethoxycarbonyl, and 9-fluorenylmethyloxycarbonyl(Fmoc); 3) aliphatic carbamate types such as tert-butyloxycarbonyl(Boc), ethoxycarbonyl, diisopropylmethoxycarbonyl, and allyloxycarbonyl;4) cyclic alkyl carbamate types such as cyclopentyloxycarbonyl andadamantyloxycarbonyl; 5) alkyl types such as triphenylmethyl and benzyl;6) trialkylsilane such as trimethylsilane; and 7) thiol containing typessuch as phenylthiocarbonyl and dithiasuccinoyl. The preferredalpha-amino protecting group is either Boc or Fmoc. Many amino acid oramino acid mimetic derivatives suitably protected for peptide synthesisare commercially, available.

The alpha-amino protecting group is cleaved prior to the coupling of thenext amino acid. When the Boc group is used, the methods of choice aretrifluoroacetic acid, neat or in dichloromethane, or HCl in dioxane. Theresulting ammonium salt is then neutralized either prior to the couplingor in situ with basic solutions such as aqueous buffers, or tertiaryamines in dichloromethane or dimethylformamide. When the Fmoc group isused, the reagents of choice are piperidine or substituted piperidinesin dimethylformamide, but any secondary amine or aqueous basic solutionscan be used. The deprotection is carried out at a temperature between 0°C. and room temperature.

Any of the amino acids or amino acid mimetics bearing side chainfunctionalities must be protected during the preparation of the peptideusing any of the above-identified groups. Those skilled in the art willappreciate that the selection and use of appropriate protecting groupsfor these side chain functionalities will depend upon the amino acid oramino acid mimetic and presence of other protecting groups in thepeptide, polypeptide or peptidomimetic. The selection of such aprotecting group is important in that it must not be removed during thedeprotection and coupling of the alpha-amino group.

For example, when Boc is chosen for the alpha-amine protection thefollowing protecting groups are acceptable: p-toluenesulfonyl (tosyl)moieties and nitro for arginine; benzyloxycarbonyl, substitutedbenzyloxycarbonyls, tosyl or trifluoroacetyl for lysine; benzyl or alkylesters such as cyclopentyl for glutamic and aspartic acids; benzylethers for serine and threonine; benzyl ethers, substituted benzylethers or 2-bromobenzyloxycarbonyl for tyrosine; p-methylbenzyl,p-methoxybenzyl, acetamidomethyl, benzyl, or t-butylsulfonyl forcysteine; and the indole of tryptophan can either be left unprotected orprotected with a formyl group.

When Fmoc is chosen for the alpha-amine protection usually tert-butylbased protecting groups are acceptable. For instance, Boc can be usedfor lysine, tert-butyl ether for serine, threonine and tyrosine, andtert-butyl ester for glutamic and aspartic acids.

Once the elongation of the peptide, polypeptide or peptidomimetic, orthe elongation and cyclization of a cyclic peptide or peptidomimetic iscompleted all of the protecting groups are removed. For the liquid phasesynthesis the protecting groups are removed in whatever manner asdictated by the choice of protecting groups. These procedures are wellknown to those skilled in the art.

When a solid phase synthesis is used to synthesize a cyclic peptide orpeptidomimetic, the peptide or peptidomimetic should be removed from theresin without simultaneously removing protecting groups from functionalgroups that might interfere with the cyclization process. Thus, if thepeptide or peptidomimetic is to be cyclized in solution, the cleavageconditions need to be chosen such that a free a-carboxylate and a freea-amino group are generated without simultaneously removing otherprotecting groups. Alternatively, the peptide or peptidomimetic may beremoved from the resin by hydrazinolysis, and then coupled by the azidemethod. Another very convenient method involves the synthesis ofpeptides or peptidomimetics on an oxime resin, followed byintramolecular nucleophilic displacement from the resin, which generatesa cyclic peptide or peptidomimetic (Osapay, Profit, and Taylor (1990)Tetrahedron Letters 43, 6121-6124). When the oxime resin is employed,the Boc protection scheme is generally chosen. Then, the preferredmethod for removing side chain protecting groups generally involvestreatment with anhydrous HF containing additives such as dimethylsulfide, anisole, thioanisole, or p-cresol at 0° C. The cleavage of thepeptide or peptidomimetic can also be accomplished by other acidreagents such as trifluoromethanesulfonic acid/trifluoroacetic acidmixtures.

Unusual amino acids used in this invention can be synthesized bystandard methods familiar to those skilled in the art (“The Peptides:Analysis, Synthesis, Biology, Vol. 5, pp. 342-449, Academic Press, NewYork (1981)). N-Alkyl amino acids can be prepared using proceduresdescribed in previously (Cheung et al., (1977) Can. J. Chem. 55, 906;Freidinger et al., (1982) J. Org. Chem. 48, 77 (1982)), which areincorporated herein by reference.

Additional synthetic procedures that can be used by one of skill in theart to synthesize the peptides, polypeptides and peptidomimeticstargeting moieties are described in PCT WO94/22910, the contents ofwhich are herein incorporated by reference.

The attachment of linking groups, L_(n), to the peptides, polypeptides,peptidomimetics, and non-peptides, Q; chelators or bonding units, C_(h),to the peptides, polypeptides, peptidomimetics, and non-peptides, Q, orto the linking groups, Ln; and peptides, polypeptides, peptidomimetics,and non-peptides bearing a fragment of the linking group to theremainder of the linking group, in combination forming the moiety,(Q)_(d)—L_(n), and then to the moiety C_(h); can all be performed bystandard techniques. These include, but are not limited to, amidation,esterification, alkylation, and the formation of ureas or thioureas.Procedures for performing these attachments can be found in Brinkley,M., Bioconjugate Chemistry 1992, 3(1), which is incorporated herein byreference.

A number of methods can be used to attach the peptides, polypeptides,peptidomimetics, and non-peptides, Q, to paramagnetic metal ion or heavyatom containing solid particles, X², by one of skill in the art of thesurface modification of solid particles. In general, the targetingmoiety Q or the combination (Q)_(d)L_(n) is attached to a coupling groupthat react with a constituent of the surface of the solid particle. Thecoupling groups can be any of a number of silanes which react withsurface hydroxyl groups on the solid particle surface, as described inco-pending U.S. patent application Ser. No. 09/356,178, now U.S. Pat.No. 6,254,852, and can also include polyphosphonates, polycarboxylates,polyphosphates or mixtures thereof which couple with the surface of thesolid particles, as described in U.S. Pat. No. 5,520,904.

A number of reaction schemes can be used to attach the peptides,polypeptides, peptidomimetics, and non-peptides, Q, to the surfactantmicrosphere, X³. These are illustrated in following reaction schemeswhere S_(f) represents a surfactant moiety that forms the surfactantmicrosphere.Acylation Reaction:

-   -   Y is a leaving group or active ester        Disulfide Coupling:        S_(f)—SH+Q—SH----------->S_(f)—S—S—Q        Sulfonamide Coupling:        S_(f)—S(═O)₂—Y+Q—NH₂----------->S_(f)—S(═O)₂—NH—Q        Reductive Amidation:        S_(f)—CHO+Q—NH₂----------->S_(f)—NH—Q        In these reaction schemes, the substituents S_(f) and Q can be        reversed as well.

The linking group L_(n) can serve several roles. First it provides aspacing group between the metal chelator or bonding moiety, C_(h), theparamagnetic metal ion or heavy atom containing solid particle, X², andthe surfactant microsphere, X³, and the one or more of the peptides,polypeptides, peptidomimetics, or non-peptides, Q, so as to minimize thepossibility that the moieties C_(h)—X, C_(h)—X¹, X², and X³, willinterfere with the interaction of the recognition sequences of Q withangiogenic tumor vasculature receptors. The necessity of incorporating alinking group in a reagent is dependent on the identity of Q, C_(h)—X,C_(h)—X¹, X², and X³. If C_(h)—X, C_(—-X) ¹, X², and X³, cannot beattached to Q without substantially diminishing its affinity for thereceptors, then a linking group is used. A linking group also provides ameans of independently attaching multiple peptides, polypeptides,peptidomimetics, and non-peptides, Q, to one group that is attached toC_(h)—X, C_(h)—X¹, X², or X³.

The linking group also provides a means of incorporating apharmacokinetic modifier into the pharmaceuticals of the presentinvention. The pharmacokinetic modifier serves to direct thebiodistibution of the injected pharmaceutical other than by theinteraction of the targeting moieties, Q, with the receptors expressedin the tumor neovasculature. A wide variety of functional groups canserve as pharmacokinetic modifiers, including, but not limited to,carbohydrates, polyalkylene glycols, peptides or other polyamino acids,and cyclodextrins. The modifiers can be used to enhance or decreasehydrophilicity and to enhance or decrease the rate of blood clearance.The modifiers can also be used to direct the route of elimination of thepharmaceuticals. Preferred pharmacokinetic modifiers are those thatresult in moderate to fast blood clearance and enhanced renal excretion.

The metal chelator or bonding moiety, C_(h), is selected to form stablecomplexes with the metal ion chosen for the particular application.Chelators or bonding moieties for diagnostic radiopharmaceuticals areselected to form stable complexes with the radioisotopes that haveimageable gamma ray or positron emissions, such as ^(99m)Tc, ⁹⁵Tc,¹¹¹In, ⁶²Cu, ⁶⁰Cu, ⁶⁴Cu, ⁶⁷Ga, ⁶⁸Ga, 86Y.

Chelators for technetium, copper and gallium isotopes are selected fromdiaminedithiols, monoamine-monoamidedithiols, triamide-monothiols,monoamine-diamide-monothiols, diaminedioximes, and hydrazines. Thechelators are generally tetradentate with donor atoms selected fromnitrogen, oxygen and sulfur. Preferred reagents are comprised ofchelators having amine nitrogen and thiol sulfur donor atoms andhydrazine bonding units. The thiol sulfur atoms and the hydrazines maybear a protecting group which can be displaced either prior to using thereagent to synthesize a radiopharmaceutical or preferably in situ duringthe synthesis of the radiopharmaceutical.

Exemplary thiol protecting groups include those listed in Greene andWuts, “Protective Groups in Organic Synthesis” John Wiley & Sons, NewYork (1991), the disclosure of which is hereby incorporated byreference. Any thiol protecting group known in the art can be used.Examples of thiol protecting groups include, but are not limited to, thefollowing: acetamidomethyl, benzamidomethyl, 1-ethoxyethyl, benzoyl, andtriphenylmethyl.

Exemplary protecting groups for hydrazine bonding units are hydrazoneswhich can be aldehyde or ketone hydrazones having substituents selectedfrom hydrogen, alkyl, aryl and heterocycle. Particularly preferredhydrazones are described in co-pending U.S. Pat. No. 5,750,088 thedisclosure of which is herein incorporated by reference in its entirety.

The hydrazine bonding unit when bound to a metal radionuclide is termeda hydrazido, or diazenido group and serves as the point of attachment ofthe radionuclide to the remainder of the radiopharmaceutical. Adiazenido group can be either terminal (only one atom of the group isbound to the radionuclide) or chelating. In order to have a chelatingdiazenido group at least one other atom of the group must also be boundto the radionuclide. The atoms bound to the metal are termed donoratoms.

Chelators for ¹¹¹In and ⁸⁶Y are selected from cyclic and acyclicpolyaminocarboxylates such as DTPA, DOTA, DO3A, 2-benzyl-DOTA,alpha-(2-phenethyl)1,4,7,10-tetraazazcyclododecane-1-acetic-4,7,10-tris(methylacetic)acid,2-benzyl-cyclohexyldiethylenetriaminepentaacetic acid,2-benzyl-6-methyl-DTPA, and6,6″-bis[N,N,N″,N″-tetra(carboxymethyl)aminomethyl)-4′-(3-amino-4-methoxyphenyl)-2,2′:6′,2″-terpyridine. Procedures for synthesizing these chelators that are notcommercially available can be found in Brechbiel, M. and Gansow, O., J.Chem. Soc. Perkin Trans. 1992, 1, 1175; Brechbiel, M. and Gansow, O.,Bioconjugate Chem. 1991, 2, 187; Deshpande, S., et. al., J. Nucl. Med.1990, 31, 473; Kruper, J., U.S. Pat. No. 5,064,956, and Toner, J., U.S.Pat. No. 4,859,777, the disclosures of which are hereby incorporated byreference in their entirety.

The coordination sphere of metal ion includes all the ligands or groupsbound to the metal. For a transition metal radionuclide to be stable ittypically has a coordination number (number of donor atoms) comprised ofan integer greater than or equal to 4 and less than or equal to 8; thatis there are 4 to 8 atoms bound to the metal and it is said to have acomplete coordination sphere. The requisite coordination number for astable radionuclide complex is determined by the identity of theradionuclide, its oxidation state, and the type of donor atoms. If thechelator or bonding unit does not provide all of the atoms necessary tostabilize the metal radionuclide by completing its coordination sphere,the coordination sphere is completed by donor atoms from other ligands,termed ancillary or co-ligands, which can also be either terminal orchelating.

A large number of ligands can serve as ancillary or co-ligands, thechoice of which is determined by a variety of considerations such as theease of synthesis of the radiopharmaceutical, the chemical and physicalproperties of the ancillary ligand, the rate of formation, the yield,and the number of isomeric forms of the resulting radiopharmaceuticals,the ability to administer said ancillary or co-ligand to a patientwithout adverse physiological consequences to said patient, and thecompatibility of the ligand in a lyophilized kit formulation. The chargeand lipophilicity of the ancillary ligand will effect the charge andlipophilicity of the radiopharmaceuticals. For example, the use of4,5-dihydroxy-1,3-benzene disulfonate results in radiopharmaceuticalswith an additional two anionic groups because the sulfonate groups willbe anionic under physiological conditions. The use of N-alkylsubstituted 3,4-hydroxypyridinones results in radiopharmaceuticals withvarying degrees of lipophilicity depending on the size of the alkylsubstituents.

Preferred technetium radiopharmaceuticals of the present invention arecomprised of a hydrazido or diazenido bonding unit and an ancillaryligand, A_(L1), or a bonding unit and two types of ancillary A_(L1) andA_(L2), or a tetradentate chelator comprised of two nitrogen and twosulfur atoms. Ancillary ligands A_(L1) are comprised of two or more harddonor atoms such as oxygen and amine nitrogen (sp³ hybridized). Thedonor atoms occupy at least two of the sites in the coordination sphereof the radionuclide metal; the ancillary ligand A_(L1) serves as one ofthe three ligands in the ternary ligand system. Examples of ancillaryligands A_(L1) include but are not limited to dioxygen ligands andfunctionalized aminocarboxylates. A large number of such ligands areavailable from commercial sources.

Ancillary dioxygen ligands include ligands that coordinate to the metalion through at least two oxygen donor atoms. Examples include but arenot limited to: glucoheptonate, gluconate, 2-hydroxyisobutyrate,lactate, tartrate, mannitol, glucarate, maltol, Kojic acid,2,2-bis(hydroxymethyl)propionic acid, 4,5-dihydroxy-1,3-benzenedisulfonate, or substituted or unsubstituted 1, 2 or 3,4hydroxypyridinones. (The names for the ligands in these examples referto either the protonated or non-protonated forms of the ligands.)

Functionalized aminocarboxylates include ligands that have a combinationof amine nitrogen and oxygen donor atoms. Examples include but are notlimited to: iminodiacetic acid, 2,3-diaminopropionic acid,nitrilotriacetic acid, N,N′-ethylenediamine diacetic acid,N,N,N′-ethylenediamine triacetic acid, hydroxyethylethylenediaminetriacetic acid, and N,N′-ethylenediamine bis-hydroxyphenylglycine. (Thenames for the ligands in these examples refer to either the protonatedor non-protonated forms of the ligands.)

A series of functionalized aminocarboxylates are disclosed by Bridgeret. al. in U.S. Pat. No. 5,350,837, herein incorporated by reference,that result in improved rates of formation of technetium labeledhydrazino modified proteins. We have determined that certain of theseaminocarboxylates result in improved yields of the radiopharmaceuticalsof the present invention. The preferred ancillary ligands A_(L1)functionalized aminocarboxylates that are derivatives of glycine; themost preferred is tricine (tris(hydroxymethyl)methylglycine).

The most preferred technetium radiopharmaceuticals of the presentinvention are comprised of a hydrazido or diazenido bonding unit and twotypes of ancillary of designated A_(L1) and A_(L2), or a diaminedithiolchelator. The second type of ancillary ligands A_(L2) are comprised ofone or more soft donor atoms selected from the group; phosphinephosphorus, arsine arsenic, imine nitrogen (sp² hybridized), sulfur (sp²hybridized) and carbon (sp hybridized); atoms which have p-acidcharacter. Ligands A_(L2) can be monodentate, bidentate or tridentate,the density is defined by the number of donor atoms in the ligand. Oneof the two donor atoms in a bidentate ligand and one of the three donoratoms in a tridentate ligand must be a soft donor atom. We havedisclosed in co-pending U.S. Pat. No. 5,744,122, and U.S. PatentApplication Ser. No. 60/013,360, now U.S. Pat. No. 5,879,659, thedisclosure of which are herein incorporated by reference in theirentirety, that radiopharmaceuticals comprised of one or more ancillaryor co-ligands A_(L2) are more stable compared to radiopharmaceuticalsthat are not comprised of one or more ancillary ligands, A_(L2); thatis, they have a minimal number of isomeric forms, the relative ratios ofwhich do not change significantly with time, and that remainsubstantially intact upon dilution.

The ligands A_(L2) that are comprised of phosphine or arsine donor atomsare trisubstituted phosphines, trisubstituted arsines, tetrasubstituteddiphosphines and tetrasubstituted diarsines. The ligands A_(L2) that arecomprised of imine nitrogen are unsaturated or aromaticnitrogen-containing, 5 or 6-membered heterocycles. The ligands that arecomprised of sulfur (Sp² hybridized) donor atoms are thiocarbonyls,comprised of the moiety C═S. The ligands comprised of carbon (sphybridized) donor atoms are isonitriles, comprised of the moiety CNR,where R is an organic radical. A large number of such ligands areavailable from commercial sources. Isonitriles can be synthesized asdescribed in European Patent 0107734 and in U.S. Pat. No. 4,988,827,herein incorporated by reference.

Preferred ancillary ligands A_(L2) are trisubstituted phosphines andunsaturated or aromatic 5 or 6 membered heterocycles. The most preferredancillary ligands A_(L2) are trisubstituted phosphines and unsaturated 5membered heterocycles.

The ancillary ligands A_(L2) may be substituted with alkyl, aryl,alkoxy, heterocycle, aralkyl, alkaryl and arylalkaryl groups and may ormay not bear functional groups comprised of heteroatoms such as oxygen,nitrogen, phosphorus or sulfur. Examples of such functional groupsinclude but are not limited to: hydroxyl, carboxyl, carboxamide, nitro,ether, ketone, amino, ammonium, sulfonate, sulfonamide, phosphonate, andphosphonamide. The functional groups may be chosen to alter thelipophilicity and water solubility of the ligands which may affect thebiological properties of the radiopharmaceuticals, such as altering thedistribution into non-target tissues, cells or fluids, and the mechanismand rate of elimination from the body.

Chelators or bonding moieties for therapeutic radiopharmaceuticals areselected to form stable complexes with the radioisotopes that have alphaparticle, beta particle, Auger or Coster-Kronig electron emissions, suchas ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu, 149 Pm, ⁹⁰Y, ²¹²Bi, ¹⁰³Pd, ¹⁰⁹Pd,¹⁵⁹Gd, ¹⁴⁰La, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁶⁵Dy, ¹⁶⁶Dy, ⁶⁷CU, ¹⁰⁵Rh,¹¹¹Ag, and ¹⁹²Ir. Chelators for rhenium, copper, palladium, platinum,iridium, rhodium, silver and gold isotopes are selected fromdiaminedithiols, monoamine-monoamidedithiols, triamide-monothiols,monoamine-diamide-monothiols, diaminedioximes, and hydrazines. Chelatorsfor yttrium, bismuth, and the lanthanide isotopes are selected fromcyclic and acyclic polyaminocarboxylates such as DTPA, DOTA, DO3A,2-benzyl-DOTA,alpha-(2-phenethyl)1,4,7,10-tetraazacyclododecane-1-acetic-4,7,10-tris(methylacetic)acid,2-benzyl-cyclohexyldiethylenetriaminepentaacetic acid,2-benzyl-6-methyl-DTPA, and6,6-bis[N,N,N″,N″-tetra(carboxyethyl)aminomethyl)-4′-(3-amino-4-methoxyphenyl)-2,2′:6′,2″-terpyridine.

Chelators for magnetic resonance imaging contrast agents are selected toform stable complexes with paramagnetic metal ions, such as Gd(III),Dy(III), Fe(III), and Mn(II), are selected from cyclic and acyclicpolyaminocarboxylates such as DTPA, DOTA, DO3A, 2-benzyl-DOTA,alpha-(2-phenethyl)1,4,7,10-tetraazacyclododecane-1-acetic-4,7,10-tris(methylacetic)acid,2-benzyl-cyclohexyldiethylenetriaminepentaacetic acid,2-benzyl-6-methyl-DTPA, and6,6″-bis[N,N,N″,N″-tetra(carboxymethyl)aminomethyl)-4′-(3-amino-4-methoxyphenyl)-2,2′:6′,2″-terpyridine.

The technetium and rhenium radiopharmaceuticals of the present inventioncomprised of a hydrazido or diazenido bonding unit can be easilyprepared by admixing a salt of a radionuclide, a reagent of the presentinvention, an ancillary ligand A_(L1), an ancillary ligand A_(L2), and areducing agent, in an aqueous solution at temperatures from 0 to 1001C.The technetium and rhenium radiopharmaceuticals of the present inventioncomprised of a tetradentate chelator having two nitrogen and two sulfuratoms can be easily prepared by admixing a salt of a radionuclide, areagent of the present invention, and a reducing agent, in an aqueoussolution at temperatures from 0 to 100° C.

When the bonding unit in the reagent of the present invention is presentas a hydrazone group, then it must first be converted to a hydrazine,which may or may not be protonated, prior to complexation with the metalradionuclide. The conversion of the hydrazone group to the hydrazine canoccur either prior to reaction with the radionuclide, in which case theradionuclide and the ancillary or co-ligand or ligands are combined notwith the reagent but with a hydrolyzed form of the reagent bearing thechelator or bonding unit, or in the presence of the radionuclide inwhich case the reagent itself is combined with the radionuclide and theancillary or co-ligand or ligands. In the latter case, the pH of thereaction mixture must be neutral or acidic.

Alternatively, the radiopharmaceuticals of the present inventioncomprised of a hydrazido or diazenido bonding unit can be prepared byfirst admixing a salt of a radionuclide, an ancillary ligand A_(L1), anda reducing agent in an aqueous solution at temperatures from 0 to 100°C. to form an intermediate radionuclide complex with the ancillaryligand A_(L1) then adding a reagent of the present invention and anancillary ligand A_(L2) and reacting further at temperatures from 0 to100° C.

Alternatively, the radiopharmaceuticals of the present inventioncomprised of a hydrazido or diazenido bonding unit can be prepared byfirst admixing a salt of a radionuclide, an ancillary ligand A_(L1), areagent of the present invention, and a reducing agent in an aqueoussolution at temperatures from 0 to 100° C. to form an intermediateradionuclide complex, and then adding an ancillary ligand A_(L2) andreacting further at temperatures from 0 to 100° C.

The technetium and rhenium radionuclides are preferably in the chemicalform of pertechnetate or perrhenate and a pharmaceutically acceptablecation. The pertechnetate salt form is preferably sodium pertechnetatesuch as obtained from commercial Tc-99m generators. The amount ofpertechnetate used to prepare the radiopharmaceuticals of the presentinvention can range from 0.1 mCi to 1 Ci, or more preferably from 1 to200 mCi. The amount of the reagent of the present invention used toprepare the technetium and rhenium radiopharmaceuticals of the presentinvention can range from 0.01 μg to 10 mg, or more preferably from 0.5μg to 200 μg. The amount used will be dictated by the amounts of theother reactants and the identity of the radiopharmaceuticals of thepresent invention to be prepared.

The amounts of the ancillary ligands A_(L1) used can range from 0.1 mgto 1 g, or more preferably from 1 mg to 100 mg. The exact amount for aparticular radiopharmaceutical is a function of identity of theradiopharmaceuticals of the present invention to be prepared, theprocedure used and the amounts and identities of the other reactants.Too large an amount of A_(L1) will result in the formation ofby-products comprised of technetium labeled A_(L1) without abiologically active molecule or by-products comprised of technetiumlabeled biologically active molecules with the ancillary ligand A_(L1)but without the ancillary ligand A_(L2). Too small an amount of A_(L1)will result in other by-products such as technetium labeled biologicallyactive molecules with the ancillary ligand A_(L2) but without theancillary ligand A_(L1), or reduced hydrolyzed technetium, or technetiumcolloid.

The amounts of the ancillary ligands A_(L2) used can range from 0.001 mgto 1 g, or more preferably from 0.01 mg to 10 mg. The exact amount for aparticular radiopharmaceutical is a function of the identity of theradiopharmaceuticals of the present invention to be prepared, theprocedure used and the amounts and identities of the other reactants.Too large an amount of A_(L2) will result in the formation ofby-products comprised of technetium labeled A_(L2) without abiologically active molecule or by-products comprised of technetiumlabeled biologically active molecules with the ancillary ligand A_(L2)but without the ancillary ligand ALI. If the reagent bears one or moresubstituents that are comprised of a soft donor atom, as defined above,at least a ten-fold molar excess of the ancillary ligand A_(L2) to thereagent of formula 2 is required to prevent the substituent frominterfering with the coordination of the ancillary ligand A_(L2) to themetal radionuclide.

Suitable reducing agents for the synthesis of the radiopharmaceuticalsof the present invention include stannous salts, dithionite or bisulfitesalts, borohydride salts, and formamidinesulfinic acid, wherein thesalts are of any pharmaceutically acceptable form. The preferredreducing agent is a stannous salt. The amount of a reducing agent usedcan range from 0.001 mg to 10 mg, or more preferably from 0.005 mg to 1mg.

The specific structure of a radiopharmaceutical of the present inventioncomprised of a hydrazido or diazenido bonding unit will depend on theidentity of the reagent of the present invention used, the identity ofany ancillary ligand A_(L1), the identity of any ancillary ligandA_(L2), and the identity of the radionuclide. Radiopharmaceuticalscomprised of a hydrazido or diazenido bonding unit synthesized usingconcentrations of reagents of <100 μg/mL, will be comprised of onehydrazido or diazenido group. Those synthesized using >1 mg/mLconcentrations will be comprised of two hydrazido or diazenido groupsfrom two reagent molecules. For most applications, only a limited amountof the biologically active molecule can be injected and not result inundesired side-effects, such as chemical toxicity, interference with abiological process or an altered biodistribution of theradiopharmaceutical. Therefore, the radiopharmaceuticals which requirehigher concentrations of the reagents comprised in part of thebiologically active molecule, will have to be diluted or purified aftersynthesis to avoid such side-effects.

The identities and amounts used of the ancillary ligands A_(L1) andA_(L2) will determine the values of the variables y and z. The a valuesof y and z can independently be an integer from 1 to 2. In combination,the values of y and z will result in a technetium coordination spherethat is made up of at least five and no more than seven donor atoms. Formonodentate ancillary ligands A_(L2), z can be an integer from 1 to 2;for bidentate or tridentate ancillary ligands A_(L2), z is 1. Thepreferred combination for monodentate ligands is y equal to 1 or 2 and zequal to 1. The preferred combination for bidentate or tridentateligands is y equal to 1 and z equal to 1.

The indium, copper, gallium, silver, palladium, rhodium, gold, platinum,bismuth, yttrium and lanthanide radiopharmaceuticals of the presentinvention can be easily prepared by admixing a salt of a radionuclideand a reagent of the present invention, in an aqueous solution attemperatures from 0 to 100° C. These radionuclides are typicallyobtained as a dilute aqueous solution in a mineral acid, such ashydrochloric, nitric or sulfuric acid. The radionuclides are combinedwith from one to about one thousand equivalents of the reagents of thepresent invention dissolved in aqueous solution. A buffer is typicallyused to maintain the pH of the reaction mixture between 3 and 10.

The gadolinium, dysprosium, iron and manganese metallopharmaceuticals ofthe present invention can be easily prepared by admixing a salt of theparamagnetic metal ion and a reagent of the present invention, in anaqueous solution at temperatures from 0 to 100° C. These paramagneticmetal ions are typically obtained as a dilute aqueous solution in amineral acid, such as hydrochloric, nitric or sulfuric acid. Theparamagnetic metal ions are combined with from one to about one thousandequivalents of the reagents of the present invention dissolved inaqueous solution. A buffer is typically used to maintain the pH of thereaction mixture between 3 and 10.

The total time of preparation will vary depending on the identity of themetal ion, the identities and amounts of the reactants and the procedureused for the preparation. The preparations may be complete, resultingin >80% yield of the radiopharmaceutical, in 1 minute or may requiremore time. If higher purity metallopharmaceuticals are needed ordesired, the products can be purified by any of a number of techniqueswell known to those skilled in the art such as liquid chromatography,solid phase extraction, solvent extraction, dialysis or ultrafiltration.

Buffers useful in the preparation of metallopharmaceuticals and indiagnostic kits useful for the preparation of said radiopharmaceuticalsinclude but are not limited to phosphate, citrate, sulfosalicylate, andacetate. A more complete list can be found in the United StatesPharmacopeia.

Lyophilization aids useful in the preparation of diagnostic kits usefulfor the preparation of radiopharmaceuticals include but are not limitedto mannitol, lactose, sorbitol, dextran, Ficoll, andpolyvinylpyrrolidine(PVP).

Stabilization aids useful in the preparation of metallopharmaceuticalsand in diagnostic kits useful for the preparation ofradiopharmaceuticals include but are not limited to ascorbic acid,cysteine, monothioglycerol, sodium bisulfite, sodium metabisulfite,gentisic acid, and inositol.

Solubilization aids useful in the preparation of metallopharmaceuticalsand in diagnostic kits useful for the preparation ofradiopharmaceuticals include but are not limited to ethanol, glycerin,polyethylene glycol, propylene glycol, polyoxyethylene sorbitanmonooleate, sorbitan monoloeate, polysorbates,poly(oxyethylene)poly(oxypropylene)poly (oxyethylene) block copolymers(Pluronics) and lecithin. Preferred solubilizing aids are polyethyleneglycol, and Pluronics.

Bacteriostats useful in the preparation of metallopharmaceuticals and indiagnostic kits useful-for the preparation of radiopharmaceuticalsinclude but are not limited to benzyl alcohol, benzalkonium chloride,chlorbutanol, and methyl, propyl or butyl paraben.

A component in a diagnostic kit can also serve more than one function. Areducing agent can also serve as a stabilization aid, a buffer can alsoserve as a transfer ligand, a lyophilization aid can also serve as atransfer, ancillary or co-ligand and so forth.

The diagnostic radiopharmaceuticals are administered by intravenousinjection, usually in saline solution, at a dose of 1 to 100 mCi per 70kg body weight, or preferably at a dose of 5 to 50 mCi. Imaging isperformed using known procedures.

The therapeutic radiopharmaceuticals are administered by intravenousinjection, usually in saline solution, at a dose of 0.1 to 100 mCi per70 kg body weight, or preferably at a dose of 0.5 to 5 mCi per 70 kgbody weight. The dosing regimen may involve a single administration ormay involve a series of fractional administrations termed fractionateddosing. The choice of dosing regimen is determine by the clinical stateof the patient, the presence of other medications and thepharmacokinetic properties of the radiopharmaceutical. Theradiopharmaceuticals of the present invention may also be used incombination therapies in which both a chemotherapeutic agent and aradiopharmaceutical of the present invention are administered in thesame or different dosing regimens. A list of acceptable chemotherapeuticagents and dosing information can be found in the United StatesPharmacopeia.

The magnetic resonance imaging contrast agents of the present inventionmay be used in a similar manner as other MRI agents as described in U.S.Pat. No. 5,155,215; U.S. Pat. No. 5,087,440; Margerstadt et al., Magn.Reson. Med., 1986, 3, 808; Runge et al., Radiology, 1988, 166, 835; andBousquet et al., Radiology, 1988, 166, 693. Generally, sterile aqueoussolutions of the contrast agents are administered to a patientintravenously in dosages ranging from 0.01 to 1.0 mmoles per kg bodyweight.

For use as X-ray contrast agents, the compositions of the presentinvention should generally have a heavy atom concentration of 1 mM to 5M, preferably 0.1 M to 2 M. Dosages, administered by intravenousinjection, will typically range from 0.5 mmol/kg to 1.5 mmol/kg,preferably 0.8 mmol/kg to 1.2 mmol/kg. Imaging is performed using knowntechniques, preferably X-ray computed tomography.

The ultrasound contrast agents of the present invention are administeredby intravenous injection in an amount of 10 to 30 μL of the echogenicgas per kg body weight or by infusion at a rate of approximately 3μL/kg/min. Imaging is performed using known techniques of sonography.

Other features of the invention will become apparent in the course ofthe following descriptions of exemplary embodiments which are given forillustration of the invention and are not intended to be limitingthereof.

EXAMPLE 1

-   Synthesis of    2-{(5-(3-(2-[(6-Hydroxycarbamoyl-7-isobutyl-8-oxo-2-oxa-9-aza-bicyclo[10.2.2]hexadeca-1(15),12(16),13-triene-10-carbonyl)-amino]-acetylamino}-propylcarbamoyl)-pyridin-2-yl]-hydrazonomethyl}-benzenesulfonic    acid

A. Preparation of[3-(2-Benzyloxycarbonylamino-acetylamino)-propyl]-carbamic Acidtert-butyl Ester

To 3 grams of (3-Amino-propyl)-carbamic acid tert-butyl ester in 15 mlof dimethylformamide was added 3 grams of N-benzyloxycarbonyl glycine,4.7 mL of N-methylmorpholine and 5.06 grams of TBTU. The reaction wascooled to 0 degrees C. for 30 minutes then allowed to stir at roomtemperature overnight. The volatiles were removed under reduced pressureand the resulting material was dissolved in ethyl acetate and washedwith 10% citric acid. The aqueous was extracted an additional two timeswith ethyl acetate, combined and washed with water, saturated aqueoussodium bicarbonate, water, brine and dried over MgSO₄. The volatileswere removed under reduced pressure and the resulting material wascrystallized from EtOAc/hexane affording 4.55 grams of the desiredproduct as a tan solid. LRMS found 388.3=(M+Na)⁺.

B. Preparation of [3-(2-Amino-acetylamino)-propyl]-carbamic Acidtert-butyl Ester

To 4.16 grams of the compound from Example 1A in 25 mL of methanol wasadded 0.5 grams of 10% Pd-C. The reaction was stirred under H₂ (balloon)for 2 hours. The reaction was filtered through a 0.45 μM PTFE filter andthe volatiles were removed under reduced pressure affording 2.5 grams ofthe desired product. LRMS found 232.3 (M+H)+⁺¹.

C. Preparation of3-{2-[(6-Benzyloxycarbamoyl-7-isobutyl-8-oxo-2-oxa-9-aza-bicyclo[10.2.2]hexadeca-1(15),12(16),13-triene-10-carbonyl)-amino}-propyl)-carbamicAcid tert-butyl Ester

To 0.25 grams of6-Benzyloxycarbamoyl-7-isobutyl-9-oxo-2-oxa-9-aza-bicyclo[10.2.2]hexadeca-1(15),12(16),13-triene-10-carboxylicacid in 10 mL of dimethylformamide was added 0.17 ml N-methylmorpholineand 0.217 grams of TUTU. After 10 minutes 0.359 grams of the compoundfrom Example 1B was added. The reaction was allowed to stir at roomtemperature overnight, then it was heated at 70 degrees C. for 30minutes. The volatiles were removed under reduced pressure and theresulting material was dissolved in EtOAc, washed with 10% aqueouscitric acid, water, saturated NaHCO₃, brine and dried over MgSO₄. Theresulting material was chromatographed on silica gel eluting with 2%MeOH/CHCl₃ affording 0.274 grams of the desired product.

D. Preparation of3-{2-[(6-Hydroxycarbamoyl-7-isobutyl-8-oxo-2-oxa-9-aza-bicyclo[10.2.2]hexadeca-1(15),12(16),13-triene-10-carbonyl)-amino}-propyl)-carbamicAcid tert-butyl Ester

To 0.035 grams of the compound from Example 1C in 5 mL of methanol wasadded 0.050 grams of 5% Pd/BaSO₄. The reaction was stirred underhydrogen (balloon) for 2 hours, then filtered through a 0.45 μM PTFEfilter and the volatiles were removed under reduced pressure affording0.031 grams of the desired compound. LRMS found 604.4 (M−H)⁻¹.

E. Preparation of7-Isobutyl-8-oxo-2-oxa-9-aza-bicyclo[10.2.2]hexadeca-1(15),12(16),13-triene-6,10-dicarboxylicacid 10-{[(3-amino-propylcarbamoyl)-methyl]-amide}6-hydroxyamideTrifluoroacetic Acid Salt

To 0.025 grams of the compound form Example 1D was added 1 mL oftrifluoroacetic acid. The reaction was stirred 1 hour and the volatileswere removed under reduced pressure affording 0.017 grams of the desiredcompound. LRMS found 506.4 (M+H)⁺¹.

F. Preparation of2-{[5-(3-{2-[(6-Hydroxycarbamoyl-7-isobutyl-8-oxo-2-oxa-9-aza-bicyclo[10.2.2]hexadeca-1(15),12(16),13-triene-10-carbonyl)-amino]-acetylamino}-propylcarbamoyl)-pyridin-2-yl]-hydrazonomethyl}-benzenesulfonicAcid

To a stirred solution of 0.050 grams of the compound from Example 1E wasadded 0.031 mL of N-methylmorpholine and 0.035 grams of6-[N″-(2-Sodio-sulfo-benzylidene)-hydrazino]-nicotinic acid2,5-dioxo-pyrrolidin-1-yl ester. The reaction was stirred at ambienttemperature overnight. Volatiles were removed under reduced pressure andthe resulting material was purified by reverse phase HPLC affording 0.08grams of the desired compound. LRMS found 807 (M−H)⁻¹.

EXAMPLE 2 Synthesis of2-{[5-(4-{[(6-Hydroxycarbamoyl-7-isobutyl-8-oxo-2-oxa-9-aza-bicyclo[10.2.2]hexadeca-1(15),12(16),13-triene-10-carbonyl)-amino]-methyl}-benzylcarbamoyl)-pyridin-2-yl]-hydrazonomethyl}-benzenesulfonicAcid

A. Preparation of (4-Aminomethyl-benzyl)carbamic Acid tert-butyl Ester

To a stirred solution of 5.3 grams of p-xylenediamine 10 in 20 mL ofdimethylformamide was added a solution of 2.12 grams ofdi-tert-butyl-dicarbonate in 50 mL of dimethylformamide by syringe pumpover 1 hour. After stirring an additional 10 minutes the volatiles wereremoved under reduced pressure and the resulting material waschromatographed on silica gel eluting with 5% MeOH/CHCl₃ affording 2grams of the desired compound. LRMS found 237.2 (M+H)⁺¹.

B. Preparation of(4-{[(6-Benzyloxycarbamoyl-7-isobutyl-8-oso-2-oxa-9-aza-bicyclo[10.2.2]hexadeca-1(15),12(16),13-triene-10-carbonyl)-amino]-methyl}-benzyl)-carbamicAcid tert-butyl Ester

To 0.20 grams of6-Benzyloxycarbamoyl-7-isobutyl-9-oxo-2-oxa-9-aza-bicyclo[10.2.2]hexadeca-1(15),12(16),13-triene-10carboxylicacid in 5 mL of dimethylformamide was added 0.18 mL ofn-methylmorpholine and 0.173 grams of TBTU. After stirring 20 minutes0.293 grams of the compound from Example 2A was added. After stirring atambient temperature overnight the reaction was heated to 80 degrees C.for 30 minutes. The volatiles were removed under reduced pressure andthe resulting material was dissolved in EtOAc, washed with 10% aqueouscitric acid, water, saturated NaHCO₃, brine and dried over MgSO₄. Thevolatiles were removed under reduced pressure affording 0.296 grams ofthe desired compound. LRMS found 699.4 (M−H)⁻¹.

C. Preparation of(4-{[(6-Hydroxycarbamoyl-7-isobutyl-8-oso-2-oxa-9-aza-bicyclo[10.2.2]hexadeca-1(15),12(16),13-triene-10-carbonyl)-amino]-methyl}-benzyl)-carbamicAcid tert-butyl Ester

To 0.275 grams of the compound from Example 2B in 20 mL of methanol wasadded 0.50 grams of pre-hydrogenated 5% pd-BaSO₄. The reaction wasstirred 3 hours under H₂ (Balloon) at which time an additional portionof 0.25 grams of 5% Pd-BaSO4 was added and the stirring was continuedfor another hour. The mixture was filtered through a 0.45 uM PTFE filterand the volatiles were removed under reduced pressure affording 0.24grams of the desired compound. LRMS found 609.4 (M−H)⁻¹.

D. Preparation of7-Isobutyl-8-oxo-2-oxa-9-aza-bicyclo[10.2.2]hexadeca-1(15),12(16),13-triene-6,10-dicarboxylicacid 10-(4-aminomethyl-benzylamice) 6-hydroxyamide Trifluoroacetic AcidSalt

To 0.225 grams of the compound from Example 2C in 5 mL of CH₂Cl₂ wasadded 2 mL of trifluoroacetic acid. The reaction was stirred one hour atambient temperature. The volatiles were removed under reduced pressureaffording the desired compound. LRMS found 509.4 (M−H)⁻¹.

E. Preparation of2-{[5-(4-{[(6-Hydroxycarbamoyl-7-isobutyl-8-oxo-2-oxa-9-aza-bicyclo[10.2.2]hexadeca-1(15),12(16),13-triene-10-carbonyl)-amino]-methyl}-benzylcarbamoyl)-pyridin-2-yl]-hydrazonomethyl}-benzenesulfonicAcid

To 0.050 grams of the compound from Example 2D in 1 mL ofdimethylformamide was added 0.031 mL of N-methylmorpholine and 0.035grams of 6-[N″-(2-Sodio-sulfo-benzylidene)-hydrazino]-nicotinic acid2,5-dioxo-pyrrolidin-1-yl ester. After stirring overnight an ambienttemperature the volatiles were removed under reduced pressure and theresulting material was purified by reverse phase HPLC affording 0.06grams the desired compound. LRMS found 814 (M+H)⁺¹.

EXAMPLE 3 Synthesis of2-[7-({N-[3-(2-{[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]carbonylamino}acetylamino)propyl]carbamoyl}methyl)-1,4,7,10-tetraaza-4,10-bis(carboxymethyl)cyclododecyl]aceticAcid

A solution of the commercially (Macrocyclics) available DOTA tri-t-butylester (1.5 mmol) and Hunig's base (6 mmol) in anhydrous DMF are treatedwith HBTU (1.25 mmol) and allowed to react for 15 min at ambienttemperatures under nitrogen.2-{[7-(N-Hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]carbonylamino)-N-(3-aminopropyl)acetamideTFA salt (1 mmol) is added to this solution and stirring is continued atambient temperatures under nitrogen for 18 h. The DMF is removed undervacuum and the resulting residue is triturated in ethyl acetate ordiethyl ether and filtered. If necessary, the crude is purified bypreparative HPLC on a C18 column using a water:ACN:0.1% TFA gradient andthe product fraction is lyophilized to give the DOTA-conjugate. The DOTAconjugate is stirred in degassed TFA at room temperature under nitrogenfor 2 h. The solution is concentrated and the resulting residue ispurified by preparative HPLC on a C18 column using a water:ACN:0.1% TFAgradient. The product fraction is lyophilized to give the titlecompound.

EXAMPLE 4 Synthesis of2-{7-[(N-{[4-({[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]-carbonylamino}methyl)phenyl]methyl}carbamoyl)methyl]-1,4,7,10-tetraaza-4,10-bis(carboxymethyl)cyclododecyl}aceticAcid

A solution of the commercially (Macrocyclics) available DOTA tri-t-butylester (1.5 mmol) and Hunig's base (6 mmol) in anhydrous DMF are treatedwith HBTU (1.25 mmol) and allowed to react for 15 min at ambienttemperatures under nitrogen.[7-(N-Hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]-N-{[4-(aminomethyl)phenyl]methyl}carboxamideTFA salt.

(1 mmol) is added to this solution and stirring is continued at ambienttemperatures under nitrogen for 18 h. The DMF is removed under vacuumand the resulting residue is triturated in ethyl acetate or diethylether and filtered. If necessary, the crude is purified by preparativeHPLC on a C18 column using a water:ACN:0.1% TFA gradient and the productfraction is lyophilized to give the DOTA-conjugate.

The DOTA conjugate is stirred in degassed TFA at room temperature undernitrogen for 2 h. The solution is concentrated and the resulting residueis purified by preparative HPLC on a C18 column using a water:ACN:0.1%TFA gradient. The product fraction is lyophilized to give the titlecompound.

EXAMPLE 5 Synthesis of2-(7-{[N-(1-{N-[3-(2-{[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3yl]carbonylamino}acetylamino)propyl]carbamoyl}-2-sulfoethyl)carbamoyl]methyl}-1,4,7,10-tetraaza-4,10-bis(carboxymethyl)cyclododecyl)aceticAcid

A. Preparation ofN-[3-(2-{[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]carbonylamino}acetylamino)propyl]-2-aminopropanesulfonicAcid

2-{([7-(N-Hydroxycarbamoyl) (3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]carbonylamino}-N-(3-aminopropyl) acetamide TFA salt (1 mmol) isdissolved in anhydrous DMF, and treated with the N-hydroxysuccinimideester (1.5 mmol) of Boc-cysteic acid (as described in Liebigs Ann. Chem.1979, 776-783) and Hunig's base. The solution is stirred at ambienttemperatures under nitrogen for 18 h, and the DMF is removed undervacuum. The resulting residue is purified by preparative HPLC on a C18column using a water:ACN:0.1% TFA gradient. The product fraction islyophilized to give a solid which is dissolved in degassed TFA andstirred at ambient temperatures for 30 min. The solution is concentratedunder vacuum, and the resulting residue is dissolved in 50% ACN andlyophilized to give the boc deprotected productN-[3-(2{[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]carbonylamino{acetylamino)propyl]-2-aminopropanesulfonicAcid.

B. Preparation of2-(7-{[N-(1-{N-[3-(2-{[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]carbonylamino}acetylamino)propyl]carbamoyl}-2-sulfoethyl)carbamoyl]methyl}-1,4,7,10-tetraaza-4,10-bis(carboxymethyl)cyclododecyl)aceticAcid.

A solution of the commercially (Macrocyclics) available DOTA tri-t-butylester (1.5 mmol) and Hunig's base (6 mmol) in anhydrous DMF are treatedwith HBTU (1.25 mmol) and allowed to react for 15 min at ambienttemperatures under nitrogen.N-[3-(2-{[7-(N-Hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]carbonylamino}acetylamino)propyl]-2-aminopropanesulfonicacid is added to this solution and stirring is continued at ambienttemperatures under nitrogen for 18 h. The DMF is removed under vacuumand the resulting residue is triturated in ethyl acetate or diethylether and filtered. If necessary, the crude is purified by preparativeHPLC on a C18 column using a water:ACN:0.1% TFA gradient and the productfraction is lyophilized to give the DOTA-conjugate.

The DOTA conjugate is stirred in degassed TFA at room temperature undernitrogen for 2 h. The solution is concentrated and the resulting residueis purified by preparative HPLC on a C18 column using a water:ACN:0.1%TFA gradient. The product fraction is lyophilized to give the titlecompound.

EXAMPLE 6 Synthesis of2-[7-({N-[1-(N-{[4-({[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]-carbonylamino}methyl)phenyl]methyl}carbamoyl)-2-sulfoethyl]carbamoyl}methyl)-1,4,7,10-tetraaza-4,10-bis(carboxymethyl)cyclododecyl]aceticAcid

A. Preparation ofN-{[4-({[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]-carbonylamino}methyl)phenyl]methyl}-2-aminopropanesulfonicAcid

[7-(N-Hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]-N-{[4-(aminomethyl)phenyl]methyl}carboxamideTFA salt (1 mmol) is dissolved in anhydrous DMF, and treated with theN-hydroxysuccinimide ester (1.5 mmol) of Boc-cysteic acid (as describedin Liebigs Ann. Chem. 1979, 776-783) and Hunig's base. The solution isstirred at ambient temperatures under nitrogen for 18 h, and the DMF isremoved under vacuum. The resulting residue is purified by preparativeHPLC on a C18 column using a water:ACN:0.1% TFA gradient. The productfraction is lyophilized to give a solid, which is dissolved in degassedTFA and stirred at ambient temperatures for 30 min. The solution isconcentrated under vacuum, and the resulting residue is dissolved in 50%ACN and lyophilized to give the boc deprotected productN-{[4-({[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]-carbonylamino}methyl)phenyl]methyl}-2-aminopropanesulfonicAcid.

B. Preparation of2-[7-((N-[1-(N-{[4-({[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]-carbonylamino}methyl)phenyl]methyl}carbamoyl)-2-sulfoethyl]carbamoyl}methyl)-1,4,7,10-tetraaza-4,10-bis(carboxymethyl)cyclododecyl]aceticAcid.

A solution of the commercially (Macrocyclics) available DOTA tri-t-butylester (1.5 mmol) and Hunig's base (6 mmol) in anhydrous DMF are treatedwith HBTU (1.25 mmol) and allowed to react for 15 min at ambienttemperatures under nitrogen.N-{[4-({[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]-carbonylamino}methyl)phenyl]methyl}-2-aminopropanesulfonicacid is added to this solution and stirring is continued at ambienttemperatures under nitrogen for 18 h. The DMF is removed under vacuumand the resulting residue is triturated in ethyl acetate or diethylether and filtered. It necessary, the crude is purified by preparativeHPLC on a C18 column using a water:ACN:0.1% TFA gradient and the productfraction is lyophilized to give the DOTA-conjugate.

The DOTA conjugate is stirred in degassed TFA at room temperature undernitrogen for 2 h. The solution is concentrated and the resulting residueis purified by preparative HPLC on a C18 column using a water:ACN:0.1%TFA gradient. The product fraction is lyophilized to give the titlecompound.

EXAMPLE 7 Synthesis of2-({2-[({N-[3-(2-{[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]carbonylamino{acetylamino)propyl]carbamoyl}methyl)(carboxymethyl)amino}ethyl){2-[bis(carboxymethyl)amino]ethyl}amino]acetic Acid

To a solution of2-{[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]carbonylamino}-N-(3-aminopropyl)acetamideTFA salt (1 mmol) in DMF (20 mL) is added triethylamine (3 mmol). Thissolution is added dropwise over 4 h to a solution ofdiethylenetriaminepentaacetic dianhydride (3 mmol) in DMF (20 mL) andmethyl sulfoxide (20 mL). The reaction mixture is then stirred for 16 h,concentrated to an oil under high vacuum and purified by preparativeHPLC to give the title compound.

EXAMPLE 8 Synthesis of2-[(2-{[(N-{[4-({[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2)hexadeca-1(15),12(16),13-trien-3-yl]-carbonylamino}methyl)phenyl]methyl}carbamoyl)methyl](carboxymethyl)amino}ethyl){2-[bis(carboxymethyl)amino]ethyl}amino]acetic Acid

To a solution of[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]-N-([4-(aminomethyl)phenyl]methyl}carboxamideTFA salt (1 mmol) in DMF (20 mL) is added triethylamine (3 mmol). Thissolution is added dropwise over 4 h to a solution ofdiethylenetriaminepentaacetic dianhydride (3 mmol) in DMF (20 mL) andmethyl sulfoxide (20 mL). The reaction mixture is then stirred for 16 h,concentrated to an oil under high vacuum and purified by preparativeHPLC to give the title compound.

EXAMPLE 9 Synthesis ofN-[3-(2-{[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]carbonylamino}acetylamino)propyl]-4,5-bis[2-(ethoxyethylthio)acetylamino]pentanamide

To a solution of 2-{([7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]carbonylamino}-N-(3-aminopropyl)acetamideTFA salt (1 mmol) and triethylamine (3 mmol) in DMF is added2,3,5,6-tetrafluorophenyl4,5-bis(S-1-ethoxyethyl-mercapto-acetamido)pentanoate (1.1 mmol), andthe reaction mixture is stirred for 18 hours. DMF is removed in vacuoand the crude residue is triturated with ethyl acetate. The product isfiltered, dried, and if necessary, further purified by preparative HPLCto give the title compound.

EXAMPLE 10 Synthesis ofN-{[4-({[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]carbonylamino}methyl)-phenyl]methyl}-4,5-bis[2-(ethoxyethylthio)acetylamino]-pentanamide

To a solution of[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]-N-{[4-(aminomethyl)phenyl]-methyl}carboxamideTFA salt (1 mmol) and triethylamine (3 mmol) in DMF is added2,3,5,6-tetrafluorophenyl4,5-bis(S-1-ethoxyethyl-mercapto-acetamido)pentanoate (1.1 mmol), andthe reaction mixture is stirred for 18 hours. DMF is removed in vacuoand the crude residue is triturated with ethyl acetate. The product isfiltered, dried, and if necessary, further purified by preparative HPLCto give the title compound.

EXAMPLE 11 Synthesis of1-(1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamino)-α,ω-dicarbonylPEG₃₄₀₀-2-{[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]carbonylamino}-N-(3-aminopropyl)acetamideConjugate

To solution of the commercially available (Shearwater Polymers)succinimidyl ester, DSPE-PEG-NHS ester (1 mmol) in 25 ml chloroform isadded 2-{[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]carbonylamino}-N-(3-aminopropyl)acetamideTFA salt (1 mmol). Sodium carbonate (1 mmol) and sodium sulfate (1 mmol)are added and the solution is stirred at room temperature under nitrogenfor 18 h. The solvent is removed in vacuo and the crude product ispurified using preparative HPLC to obtain the title compound.

EXAMPLE 12 Synthesis of1-(1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamino)-α,ω-dicarbonylPEG₃₄₀₀-[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]-N-{([4-(aminomethyl)phenyl]methyl}carboxamideConjugate

To solution of the commercially available (Shearwater Polymers)succinimidyl ester, DSPE-PEG-NHS ester (1 mmol) in 25 ml chloroform isadded [7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]-N-{[4-(aminomethyl)phenyl]methyl}carboxamideTFA salt (1 mmol). Sodium carbonate (1 mmol) and sodium sulfate (1 mmol)are added and the solution is stirred at room temperature under nitrogenfor 18 h. The solvent is removed in vacuo and the crude product ispurified using preparative HPLC to obtain the title compound.

EXAMPLES 13 and 14 Synthesis of ^(99m)Tc Complexes

To a lyophilized vial containing 4.84 mg TPPTS, 6.3 mg tricine, 40 mgmannitol, succinic acid buffer, pH 4.8, and 0.1% Pluronic F-64surfactant, was added 0.75-1.1 mL sterile water for injection, 0.2-0.45mL (20-40 μg) of the compounds of Examples 1 and 2, respectively, indeionized water or 50% aqueous ethanol, and 0.2-0.4 mL of^(99m)TcO₄-(50-120 mCi) in saline. The reconstituted kit was heated in a100° C. water bath for 10-15 minutes, and was allowed to cool 10 minutesat room temperature. A sample was then analyzed by HPLC.

HPLC Method for Example 13

-   Column: Zorbax C18, 25 cm×4.6 mm-   Flow rate: 1.0 mL/min-   Solvent A: 10 mM sodium phosphate buffer, pH 6.0-   Solvent B: 100% CH₃CN

t (min) 0 20 21 30 31 40 % Solvent B 0 25 75 75 0 0HPLC Method for Example 14

-   Column: Zorbax C18, 25 cm×4.6 mm-   Flow rate: 1.0 mL/min-   Solvent A: 10 mM sodium phosphate buffer, pH 6.0-   Solvent B: 100% CH₃CN

t (min) 0 20 21 26 27 40 % Solvent B 0 25 75 75 0 0

Ret. Time Example # Reagent Ex. # (min) % Yield 13 1 7.8 79 14 2 16.7 81

EXAMPLE 15 Synthesis of2-[2-({5-[N-(5-(N-hydroxycarbamoyl)(5R)-5-{3-[4-(3,4-dimethoxyphenoxy)phenyl]-3-methyl-2-oxopyrrolidinyl}pentyl)carbamoyl](2-pyridyl)}amino)(1Z)-2-azavinyl]benzenesulfonicAcid

The title compound can be synthesized as shown in Scheme I from thestarting materials described in the patent applications incorporated byreference above.

EXAMPLE 16 Synthesis of2-(2-{[5-(N-{3-[3-(N-hydroxycarbamoyl)(4S)-4-({4-[(4-methylphenyl)methoxy]piperidyl}carbonyl)piperidyl]-3-oxopropyl}carbamoyl)(2-pyridyl)]amino}(1Z)-2-azavinyl)benzenesulfonicAcid

The title compound can be synthesized as shown in Scheme II from thestarting materials described in patent applications incorporated byreference above.

EXAMPLE 17 Synthesis of N-BOC-Glycine-(3-carbobenzyloxyamido)propylamide

Di-isopropylethylamine (7.0 mL, 40 mmol) was added to a suspension ofN-t-butyloxycarbonylglycine N-hydroxysuccinimide ester (5.56 g, 20 mmol)and N-carbobenzyloxy-1,3-diaminopropane hydrochloride (5.0 g, 20 mmol)in dichloromethane (50 ml). The solution became clear over severalminutes. After 30 minutes, additional of N-t-butyloxycarbonylglycineN-hydroxysuccinimide ester (0.275 g, 1 mmol) was added. The solution wasextracted with water, followed by saturated aqueous NaHCO₃, then by 0.5N HCl. The dichloromethane solution was filtered through a short columnof Na₂SO₄ and evaporated in vacuo to obtain 4.1 g (56%) ofN-BOC-Glycine-(3-carbobenzyloxyamido)propylamide. MS: m/e=366 (M+H⁺),310 (M−C₄H₉+H⁺), 266 (M−BOC+H⁺).

Synthesis of Glycine-(3-carbobenzyloxyamido)propylamide:

N-BOC-Glycine-(3-carbobenzyloxyamido)propylamide (260 mg, 0.71 mmol) wasdissolved in dichloromethane (3 mL) and trifluoroacetic acid (0.5 mL)added. After 20 minutes, the solution was evaporated in vacuo, dissolvedin dichloromethane (2 mL) and evaporated in vacuo to obtain the crudeproduct which was used directly in the next reaction. MS: m/e=266(M+H⁺).

Synthesis of 17a:

Di-isopropylethylamine (0.25 mL, 1.4 mmol) was added to a mixture of 1(300 mg, 0.69 mmol) and HBTU (270 mg, 0.71 mmol) in dichloromethane (5mL). Dimethylformamide (1 mL) was added to obtain a clear solution.After 30 minutes, a solution ofGlycine-(3-carbobenzyloxyamido)propylamide (˜0.71 mmol) anddi-isopropylethyl amine (0.25 mL, 1.4 mmol) in dichloromethane (1 mL)was added. The reaction mixture was extracted twice with 0.5 N HCl, onceeach with saturated aqueous NaCl, 1.0 N NaOH, saturated aqueous NaCl,and saturated aqueous NaHCO₃. The dichloromethane solution was filteredthrough a short column of Na₂SO₄ and evaporated in vacuo to obtain crude17b (599 mg, 127%). MS: m/e 681 (M+H₊).

Synthesis of 17b:

Trifluoroacetic acid (1 mL) was added to a solution of 17a (599 mg) indichloromethane (5 mL) and allowed to stand at room temperatureovernight. The solution was evaporated in vacuo, dissolved indichloromethane (2 mL) and evaporated in vacuo to obtain 3 (714 mg). MS:m/e=625 (M+H⁺).

Synthesis of 17c

A mixture of 3 (˜0.3 g, ˜0.5 mmol) and 10% Pd/C (25 mg) in ethanol (5mL) was stirred under hydrogen (1 atm) for 2.5 hours. Disappearance of17b was accompanied by the appearance of two peaks in the HPLC-MSchromatogram, both of which exhibited base peaks at m/e=491 amu,consistent with (M+H⁺) for the loss of carbobenzyloxy group from 26b.The reaction mixture was filtered through Celite and evaporated in vacuoto obtain 17c.

Synthesis of 17d

A mixture ofN,N-bis[2-bis(1,1′-dimethylethoxy)-2-oxoethyl]-amino]ethyl]glycine (487mg, 0.788 mmol), HBTU (288 mg, 0.760 mmol)and di-isopropylethyl amine(0.4 mL, 2.3 mmol) in dimethylformamide (4 mL) was stirred at roomtemperature. A solution of 4 (−0.5 mmol) in dimethylformamide (2 mL) wasadded in one portion. After 2 hours, ˜⅔ of the solution was removed,partitioned between dichloromethane and 0.5 M HCl. The organic phaseextracted once with 0.5 M HCl, then with saturated aqueous NaCl,filtered through a column of Na₂SO₄, and evaporated in vacuo to obtaincrude 17d. MS: m/e 1090 (M+H⁺), 546 (M+2H)⁺²

Synthesis of 17e

The remaining ⅓ of the reaction mixture 17d was treated with HBTU (75mg, 0.20 mmol) and allowed to stir for 20 minutes. A solution preparedfrom hydroxylamine hydrochloride (50 mg, 0.70 mmol) anddi-isopropylethylamine (0.15 mL, 0.86 mmol) in dimethylformamide (0.5mL) was added in one portion. The reaction mixture was partitionedbetween dichloromethane and 0.5 M HCl. The organic phase was extractedwith saturated aqueous NaCl, then with saturated aqueous NaHCO₃. TheNaHCO₃ phase was back-extracted with dichloromethane, the combinedorganic extracts filtered through a column of Na₂SO₄, and evaporated invacuo. The crude product was purified by reverse-phase HPLC to obtain 14mg of 27e. MS: m/e 1105 (M+H⁺), 553 (M+2H)⁺²

Synthesis of 17f

A solution of 17e in trifluoroacetic acid (0.5 mL) and dichloromethane(2 mL) was allowed to stand at room temperature overnight. The solutionwas evaporated in vacuo, dissolved in acetonitrile-water and purified byreverse-phase HPLC to obtain 3.4 mg of 17f. MS: m/e 881 (M+H⁺), 441(M+2H)⁺².

UTILITY

The pharmaceuticals of the present invention are useful for imaging ofprocesses involving the degradation of the extracellular matrixincluding cancer, diabetic retinopathy and macular degeneration. Theradiopharmaceuticals of the present invention comprised of a gammaemitting isotope are useful for imaging of these pathological processesand the radiopharmaceuticals comprised of a beta particle, alphaparticle or Auger electron emitting isotope are useful for treatingthese pathologies.

The compounds of the present invention comprised of one or moreparamagnetic metal ions selected from gadolinium, dysprosium, iron, andmanganese, are useful as contrast agents for magnetic resonance imaging(MRI) of cardiovascular pathological processes involving extracellularmatrix degradation.

The compounds of the present invention comprised of one or more heavyatoms with atomic number of 20 or greater are useful as X-ray contrastagents for X-ray imaging of cardiovascular pathological processesinvolving extracellular matrix degradation.

The compounds of the present invention comprised of an echogenic gascontaining surfactant microsphere are useful as ultrasound contrastagents for sonography of cardiovascular pathological processes involvingextracellular matrix degradation.

Representative compounds of the present invention were tested in the oneor more of the following in vitro assays and were found to be active.

Matrix Metalloproteinase Assays for MMP-1 (collagenase-1), MMP-2(gelatinase A), MMP-3 (stromelysin-1), MMP-8 (collagenase-2), MMP-9(gelatinase B), MMP-13 (collagenase-3), MMP-14(membrane type 1 MMP),MMP-15 (membrane type 2 MMP), and MMP-16 (membrane type 3 MMP).

A. Reagents

-   1. MCA peptide substrate: Mca-Pro-Leu-Gly-Leu-Dpa-Ala-NH2 (SEQ ID    NO:1). Peptide stocks are stored at −70 C in DMSO at 20 mM. Dilute    peptide in 1× reaction buffer to a working concentration of 14 uM on    day of use.-   2. Enzyme buffer. 50 mM Tricine, 0.05% Brij-35, 400 mM NaCl, 10 mM    CaCl2, 0.02% NaN3, pH 7.5.-   3. Reaction buffer. 50 mM Tricine, 10 mM CaCl2, 0.02% NaN3, pH 7.5-   4. Compounds. Stock compounds are at 10 mM in DMSO. Dilutions are    done in buffer.-   5. Plates. microfluor W flat bottom plates (Dynex Inc. Cat.#7905).    B. Assay-   1. To 96 well fluorescent assay plates add 2 uL of DMSO control or    compound dilutions to wells.-   2. Add 20 uL of EDTA (0.5M) to each quench well.-   3. Add 50 uL of enzyme at the appropriate concentration.-   4. Add 150 uL of the MCA peptide at final concentration of 10 UM.-   5. Incubate each plate for 1 hour at room temperature on an orbital    shaker.-   6. Add 20 uL of EDTA (0.5 M) to each test well to quench the    reaction.-   7. Read each plate at 330 nm excitation, 440 nm emission (Dynx plate    reader).-   8. Subtract each quench value from the corresponding test value.-   9. % inhibition =100×(sample fluorescence/control fluorescence)×100.    TACE Assay    A. Reagents-   1. MCA Peptide substrate: Mca-PLAQAV(Dpa)RSSSR-NH2 (SEQ ID NO:2).    Peptide stocks are stored at −70 C in DMSO at 20 mM. Dilute peptide    stock in reaction buffer to a working concentration of 20 uM on day    of use.-   2. Reaction buffer. 50 mM Tricine, 100 mM NaCl, 10 mM CaCl2, 1 mM    ZnCl12, pH 7.5.-   3. Compounds. Stock compounds are at 10 mM in DMSO-   4. Plates. black Packard Optiplate (Cat.# HTRF-96)-   5. Cytofluor Multi-well Plate Reader (Series 4000)    B. Assay-   1. Initiate assay by adding 2 nM TACE to buffered solutions    containing 10 μM MCA peptide substrate in the presence of increasing    concentrations of compound.-   2. Add 20 uL of EDTA (0.5M) to each quench well.-   3. Total volume is 300 uL in each well.-   4. Incubate the reaction mixtures for 1 hour at 28 C on an orbital    shaker.-   5. Add 20 uL of EDTA (0.5M) to each test well to quench the    reaction.-   6. Read each plate at 330 nM excitation, 395 nm emission-   7. Subtract each quench value from the corresponding test value.-   8. % inhibition=100−(sample fluorescence/control fluorescence)×100    Oncomouse® Imaging

The study involves the use of the c-Neu Oncomouse® and FVB micesimultaneously as controls. The mice are anesthetized with sodiumpentobarbital and injected with approximately 0.5 mCi ofradiopharmaceutical. Prior to injection, the tumor locations on eachOncomouse® are recorded and tumor size measured using calipers. Theanimals are positioned on the camera head so as to image the anterior orposterior of the animals. 5 Minute dynamic images are acquired seriallyover 2 hours using a 256×256 matrix and a zoom of 2×. Upon completion ofthe study, the images are evaluated by circumscribing the tumor as thetarget region of interest (ROI) and a background site in the neck areabelow the carotid salivary glands.

This model can also be used to assess the effectiveness of theradiopharmaceuticals of the present invention comprised of a beta, alphaor Auger electron emitting isotope. The radiopharmaceuticals areadministered in appropriate amounts and the uptake in the tumors can bequantified either non-invasively by imaging for those isotopes with acoincident imageable gamma emission, or by excision of the tumors andcounting the amount of radioactivity present by standard techniques. Thetherapeutic effect of the radiopharmaceuticals can be assessed bymonitoring the rate of growth of the tumors in control mice versus thosein the mice administered the radiopharmaceuticals of the presentinvention.

This model can also be used to assess the compounds of the presentinvention comprised of paramagnetic metals as MRI contrast agents. Afteradministration of the appropriate amount of the paramagnetic compounds,the whole animal can be placed in a commercially available magneticresonance imager to image the tumors. The effectiveness of the contrastagents can be readily seen by comparison to the images obtain fromanimals that are not administered a contrast agent.

This model can also be used to assess the compounds of the presentinvention comprised of heavy atoms as X-ray contrast agents. Afteradministration of the appropriate amount of the X-ray absorbingcompounds, the whole animal can be placed in a commercially availableX-ray imager to image the tumors. The effectiveness of the contrastagents can be readily seen by comparison to the images obtain fromanimals that are not administered a contrast agent.

This model can also be used to assess the compounds of the presentinvention comprised of an echogenic gas containing surfactantmicrosphere as ultrasound contrast agents. After administration of theappropriate amount of the echogenic compounds, the tumors in the animalcan be imaging using an ultrasound probe held proximate to the tumors.The effectiveness of the contrast agents can be readily seen bycomparison to the images obtain from animals that are not administered acontrast agent.

Rabbit Matrigel Model

This model was adapted from a matrigel model intended for the study ofangiogenesis in mice. Matrigel (Becton & Dickinson, USA) is a basementmembrane rich in laminin, collagen IV, entactin, HSPG and other growthfactors. When combined with growth factors such as bFGF [500 ng/ml] orVEGF [2 μg/ml] and injected subcutaneously into the mid-abdominal regionof the mice, it solidifies into a gel and stimulates angiogenesis at thesite of injection within 4-8 days. In the rabbit model, New ZealandWhite rabbits (2.5-3.0 kg) are injected with 2.0 ml of matrigel, plus 1μg bFGF and 4 μg VEGF. The radiopharmaceutical is then injected 7 dayslater and the images obtained.

This model can also be used to assess the effectiveness of theradiopharmaceuticals of the present invention comprised of a beta, alphaor Auger electron emitting isotope. The radiopharmaceuticals areadministered in appropriate amounts and the uptake at the angiogenicsites can be quantified either non-invasively by imaging for thoseisotopes with a coincident imageable gamma emission, or by excision ofthe angiogenic sites and counting the amount of radioactivity present bystandard techniques. The therapeutic effect of the radiopharmaceuticalscan be assessed by monitoring the rate of growth of the angiogenic sitesin control rabbits versus those in the rabbits administered theradiopharmaceuticals of the present invention.

This model can also be used to assess the compounds of the presentinvention comprised of paramagnetic metals as MRI contrast agents. Afteradministration of the appropriate amount of the paramagnetic compounds,the whole animal can be placed in a commercially available magneticresonance imager to image the angiogenic sites. The effectiveness of thecontrast agents can be readily seen by comparison to the images obtainfrom animals that are not administered a contrast agent.

This model can also be used to assess the compounds of the presentinvention comprised of heavy atoms as X-ray contrast agents. Afteradministration of the appropriate amount of the X-ray absorbingcompounds, the whole animal can be placed in a commercially availableX-ray imager to image the angiogenic sites. The effectiveness of thecontrast agents can be readily seen by comparison to the images obtainfrom animals that are not administered a contrast agent.

This model can also be used to assess the compounds of the presentinvention comprised of an echogenic gas containing surfactantmicrosphere as ultrasound contrast agents. After administration of theappropriate amount of the echogenic compounds, the angiogenic sites inthe animal can be imaging using an ultrasound probe held proximate tothe tumors. The effectiveness of the contrast agents can be readily seenby comparison to the images obtain from animals that are notadministered a contrast agent.

Canine Spontaneous Tumor Model

Adult dogs with spontaneous mammary tumors were sedated with xylazine(20 mg/kg)/atropine (1 ml/kg). Upon sedation the animals were intubatedusing ketamine (5 mg/kg)/diazepam (0.25 mg/kg) for full anethesia.Chemical restraint was continued with ketamine (3 mg/kg)/xylazine (6mg/kg) titrating as necessary. If required the animals were ventilatedwith room air via an endotrachael tube (12 strokes/min, 25 ml/kg) duringthe study. Peripheral veins were catheterized using 20G I.V. catheters,one to serve as an infusion port for compound while the other forexfusion of blood samples. Heart rate and EKG were monitored using acardiotachometer (Biotech, Grass Quincy, Mass.) triggered from a lead IIelectrocardiogram generated by limb leads. Blood samples are generallytaken at −10 minutes (control), end of infusion, (1 minute), 15 min, 30min, 60 min, 90 min, and 120 min for whole blood cell number andcounting. Radiopharmaceutical dose was 300 μCi/kg adminitered as an i.v.bolus with saline flush. Parameters were monitored continuously on apolygraph recorder (Model 7E Grass) at a paper speed of 10 mm/min or 10mm/sec.

Imaging of the laterals were for 2 hours with a 256×256 matrix, no zoom,5 minute dynamic images. A known source is placed in the image field(20-90 μCi) to evaluate region of interest (ROI) uptake. Images werealso acquired 24 hours post injection to determine retention of thecompound in the tumor. The uptake is determined by taking the fractionof the total counts in an inscribed area for ROI/source and multiplyingthe known μCi. The result is μCi for the ROI.

This model can also be used to assess the effectiveness of theradiopharmaceuticals of the present invention comprised of a beta, alphaor Auger electron emitting isotope. The radiopharmaceuticals areadministered in appropriate amounts and the uptake in the tumors can bequantified either non-invasively by imaging for those isotopes with acoincident imageable gamma emission, or by excision of the tumors andcounting the amount of radioactivity present by standard techniques. Thetherapeutic effect of the radiopharmaceuticals can be assessed bymonitoring the size of the tumors over time. This model can also be usedto assess the compounds of the present invention comprised ofparamagnetic metals as MRI contrast agents. After administration of theappropriate amount of the paramagnetic compounds, the whole animal canbe placed in a commercially available magnetic resonance imager to imagethe tumors. The effectiveness of the contrast agents can be readily seenby comparison to the images obtain from animals that are notadministered a contrast agent.

This model can also be used to assess the compounds of the presentinvention comprised of heavy atoms as X-ray contrast agents. Afteradministration of the appropriate amount of the X-ray absorbingcompounds, the whole animal can be placed in a commercially availableX-ray imager to image the tumors. The effectiveness of the contrastagents can be readily seen by comparison to the images obtain fromanimals that are not administered a contrast agent.

This model can also be used to assess the compounds of the presentinvention comprised of an echogenic gas containing surfactantmicrosphere as ultrasound contrast agents. After administration of theappropriate amount of the echogenic compounds, the tumors in the animalcan be imaging using an ultrasound probe held proximate to the tumors.The effectiveness of the contrast agents can be readily seen bycomparison to the images obtain from animals that are not administered acontrast agent.

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise that as specifically describedherein.

1. A radiopharmaceutical comprising a compound and a cytotoxicradioisotope; wherein said compound comprises: i) 1-10 targetingmoieties; ii) a chelator, and iii) 0-1 linking groups between thetargeting moiety and chelator; wherein the targeting moiety is a matrixmetalloproteinase inhibitor having an inhibitory constant K_(i) of <100nM of the formula (Ib);

wherein, R⁸ is independently at each occurrence OH or phenyl, optionallysubstituted with a bond to the linking group, provided that when R⁸ isphenyl, R¹⁰ is —C(═O)—CHR¹²—NH—(CH₃)—COOH; R⁹ and R^(9′) areindependently H, C₁₋₆ alkyl optionally substituted with a bond to thelinking group, or are taken together with the carbon atom to which R⁹and R^(9′) are attached to form a 5-7 atom saturated, partiallyunsaturated or aromatic ring system containing 0-3 heteroatoms selectedfrom O, N, SO₂ and S, said ring system substituted with R⁶ andoptionally substituted with a bond to the linking group; R¹⁰ and R¹¹ areindependently H, or C₁₋₆ alkyl optionally substituted with a bond tolinking group, or are taken together with the nitrogen atom to whichthey are attached to form a 5-7 atom saturated, partially unsaturated oraromatic ring system containing 0-2 additional heteroatoms selected fromO, N, SO₂ and S, said ring system optionally substituted a bond to thelinking group; or alternatively, R⁹ and R¹⁰ are taken together with thenitrogen atom and carbon atom to which they are attached to form a 5-7atom saturated, partially unsaturated or aromatic ring system containing0-2 additional heteroatoms selected from O, N, SO₂ and S, said ringsystem optionally substituted with a bond to the linking group; and R¹²is independently C₁₋₂₀ alkyl.
 2. A radiopharmaceutical according toclaim 1, comprising 1-5 targeting moieties.
 3. A radiopharmaceuticalaccording to claim 1, comprising one targeting moiety.
 4. Aradiopharmaceutical according to claim 1, wherein the linking group isof the formula:((W¹)_(h)—(CR¹³R¹⁴)_(g))_(x)—(Z)_(k)—((CR^(13a)R^(14a))_(g)′-(W²)_(h)′)_(x)′;W¹ is C(═O)NR¹⁵; R¹⁵ is H,═O, COOH, SO₃H, PO₃H, C₁-C₅ alkyl substitutedwith 0-3 R¹⁶, aryl substituted with 0-3 R¹⁶, benzyl substituted with 0-3R¹⁶, C₁-C₅ alkoxy substituted with 0-3 R¹⁶, NHC(═O)R¹⁷, C(═O)NHR¹⁷,NHR¹⁷, R¹⁷, and a bond to the chelator; R¹⁶ is independently selected ateach occurrence from the groups: a bond to the chelator, COOR¹⁷, NHR¹⁷,NHC(═O)R¹⁷, OH, NHR¹⁷, SO₃H, PO₃H, —PO₃H₂, —OSO₃H, aryl substituted with0-3 R¹⁷, C₁₋₅ alkyl substituted with 0-1 R¹⁸, C₁₋₅ alkoxy substitutedwith 0-1 R¹⁸, and 5-10 membered heterocyclic ring system containing 1-4heteroatoms independently selected from N, S, and O and substituted with0-3 R¹⁷; R¹⁷ is independently selected at each occurrence from thegroup: H, alkyl substituted with 0-1 R¹⁸, aryl substituted with 0-1 R¹⁸,a 5-10 membered heterocyclic ring system containing 1-4 heteroatomsindependently selected from N, S, and O and substituted with 0-1 R¹⁸,C₃₋₁₀ cycloalkyl substituted with 0-1 R¹⁸, polyalkylene glycolsubstituted with 0-1 R¹⁸, carbohydrate substituted with 0-1 R¹⁸,cyclodextrin substituted with 0-1 R¹⁸, amino acid substituted with 0-1R¹⁸, polycarboxyalkyl substituted with 0-1 R¹⁸, polyazaalkyl substitutedwith 0-1 R¹⁸, peptide substituted with 0-1 R¹⁸, wherein the peptide iscomprised of 2-10 amino acids, 3,6-O-disulfo-B-D-galactopyranosyl,bis(phosphonomethyl)glycine, and a bond to the chelator; R¹⁸ is a bondto the chelator; h is 1; g is 3; R¹³ and R¹⁴ are independently H; x is1; k is 0; g′ is 0; h′ is 1; W² is NH; and x′ is
 1. 5. Aradiopharmaceutical according to claim 1, wherein the linking group isof the formula:((W¹)_(h)—(CR¹³R¹⁴)_(g))_(x)—(Z)_(k)—((CR^(13a)R^(14a))_(g′)—(W²)_(h′))_(x′;)x is 0; k is 1; Z is aryl substituted with 0-3 R¹⁶; R¹⁶ is independentlyselected at each occurrence from the group: a bond to the chelator,COOR¹⁷, C(═O)NHR¹⁷, NHC(═O)R¹⁷, OH, NHR¹⁷, SO₃H, PO₃H, —OPO₃H₂, —OSO₃H,aryl substituted with 0-3 R¹⁷, C₁₋₅ alkyl substituted with 0-1 R¹⁸, C₁₋₅alkoxy substituted with 0-1 R¹⁸, and a 5-10 membered heterocyclic ringsystem containing 1-4 heteroatoms independently selected from N, S, andO and substituted with 0-3 R¹⁷; R¹⁷ is independently selected at eachoccurrence from the group; H, alkyl substituted with 0-1 R¹⁸, arylsubstituted with 0-1 R¹⁸, a 5-10 membered heterocyclic ring systemcontaining 1-4 heteroatoms independently selected from N, S, and O andsubstituted with 0-1 R¹⁸, C₃₋₁₀ cycloalkyl substituted with 0-1 R¹⁸,polyalkylene glycol substituted with 0-1 R¹⁸, carbohydrate substitutedwith 0-1 R¹⁸, cyclodextrin substituted with 0-1 R¹⁸, amino acidsubstituted with 0-1 R¹⁸, polycarboxyalkyl substituted with 0-1 R¹⁸,polyazaalkyl substituted with 0-1 R¹⁸, peptide substituted with 0-1 R¹⁸,wherein the peptide is comprised of 2-10 amino acids,3,6-O-disulfo-B-D-galactopyranosyl, bis(phosphonomethyl)glycine, and abond to the chelator; R¹⁸ is a bond to the chelator; g′ is 1; W² is NH;R^(13a) and R^(14a) are independently H; h′ is 1; and x′ is
 1. 6. Aradiopharmaceutical according to claim 1, wherein the linking group isof the formula:((W¹)_(h)—(CR¹³R¹⁴)_(g))_(x)—(Z)_(k)—((CR^(13a)R^(14a))_(g′)—(W²)_(h′))_(x′;)W¹ is C(═O)NR¹⁵; R¹⁵ is H, ═O, COOH, SO₃H, PO₃H, C₁-C₅ alkyl substitutedwith 0-3 R¹⁶, aryl substituted with 0-3 R¹⁶, benzyl substituted with 0-3R¹⁶, C₁-C₅ alkoxy substituted with 0-3 R¹⁶, NHC(═O)R¹⁷, C(═O)NHR¹⁷,NHR¹⁷, R¹⁷, and a bond to the chelator; R¹⁶ is independently selected ateach occurrence from the group; a bond to the chelator, COOR¹⁷,C(═O)NHR¹⁷, NHC(═O)R¹⁷, OH, NHR¹⁷, SO₃H, PO₃H, —OPO₃H₂, —OSO₃H, arylsubstituted with 0-3 R¹⁷, C₁₋₅ alkyl substituted with 0-1 R¹⁸, C₁₋₅alkoxy substituted with 0-1 R¹⁸, and a 5-10 membered heterocyclic ringsystem containing 1-4 heteroatoms independently selected from N, S, andO and substituted with 0-3 R¹⁷; R¹⁷ is independently selected at eachoccurrence from the group; H, alkyl substituted with 0-1 R¹⁸, arylsubstituted with 0-1 R¹⁸, a 5-10 membered heterocyclic ring systemcontaining 1-4 heteroatoms independently selected from N, S, and O andsubstituted with 0-1 R¹⁸, C₃₋₁₀ cycloalkyl substituted with 0-1 R¹⁸,polyalkylene glycol substituted with 0-1 R¹⁸, carbohydrate substitutedwith 0-1 R¹⁸, cyclodextrin substituted with 0-1 R¹⁸, amino acidsubstituted with 0-1 R¹⁸, polycarboxyalkyl substituted with 0-1 R¹⁸,polyazaalkyl substituted with 0-1 R¹⁸, peptide substituted with 0-1 R¹⁸,wherein the peptide is comprised of 2-10 amino acids3,6-O-disulfo-B-D-galactopyranosyl, bis(phosphonomethyl)glycine, and abond to the chelator; R¹⁸ is a bond to the chelator; h is 1; g is 2; R¹³and R¹⁴ are independently H; x is 1; k is 0; g′ is 1; R^(13a) andR^(14a) are independently H; or C₁₋₅ alkyl substituted with 0-3 R¹⁶; R¹⁶is SO₃H; W² is NHC(═O) or NH; h′ is 1; and x′ is
 2. 7. Aradiopharmaceutical according to claim 1, wherein the linking group isof the formula:(W¹)_(h)—(CR¹³R¹⁴)_(g))_(x)—(Z)_(k)—((CR^(13a)R^(14a))_(g)—(W²)_(h′x))_(x′;)x is 0; k is 0; R^(13a) and R^(14a) are independently H; or C₁₋₅ alkylsubstituted with 0-3 R¹⁶; R¹⁶ is independently selected at eachoccurrence from the group; a bond to the chelator, COOR¹⁷, C(═O)NHR¹⁷,NHC(═O)R¹⁷, OH, NHR¹⁷, SO₃H, PO₃H, —OPO₃H₂, —OSO₃H, aryl substitutedwith 0-3 R¹⁷, C₁₋₅ alkyl substituted with 0-1 R¹⁸, C₁₋₅ alkoxysubstituted with 0-1 R¹⁸, and a 5-10 membered heterocyclic ring systemcontaining 1-4 heteroatoms independently selected from N, S, and O andsubstituted with 0-3 R¹⁷; R¹⁷ is independently selected at eachoccurrence from the group: H, alkyl substituted with 0-1 R¹⁸, arylsubstituted with 0-1 R¹⁸, a 5-10 membered heterocyclic ring systemcontaining 1-4 heteroatoms independently selected from N, S, and O andsubstituted with 0-1 R¹⁸, C₃₋₁₀ cycloalkyl substituted with 0-1 R¹⁸,polyalkylene glycol substituted with 0-1 R¹⁸, carbohydrate substitutedwith 0-1 R¹⁸, cyclodextrin substituted with 0-1 R¹⁸, amino acidsubstituted with 0-1 R¹⁸, polycarboxyalkyl substituted with 0-1 R¹⁸,polyazaalkyl substituted with 0-1 R¹⁸, peptide substituted with 0-1 R¹⁸,wherein the peptide is comprised of 2-10 amino acids,3,6-O-disulfo-B-D-galactopyranosyl, bis(phosphonomethyl)glycine, and abond to the chelator; R¹⁸ is a bond to the chelator; g′ is 3; h′ is 1;W² is NH; and x′ is
 1. 8. A radiopharmaceutical according to claim 1,wherein the linking group is of the formula:((W¹)_(h)—(CR¹³R¹⁴)_(g))_(x)—(Z)_(k)—((CR^(13a)R^(14a))_(g′)—(W²)_(h′))_(x′;)W¹ is C═O; h is 0, 1, or 2; g is 2; R¹³ and R¹⁴ are H; x is 0, 1, 2, 3,4, or 5; k is 0; g′ is 0; h′ is 1; W² is NH; and x′ is
 1. 9. Aradiopharmaceutical according to claim 1, wherein the linking group isabout.
 10. A radiopharmaceutical comprising: a cytotoxic radioisotopeand a compound selected from the group consisting of:2-{[5-(3-{2-[(6-Hydroxycarbamoyl-7-isobutyl-8-oxo-2-oxa-9-aza-bicyclo[10.2.2]hexadeca-1(15),12(16),13-triene-10-carbonyl)-amino]acetylamino}-propylcarbamoyl)-pyridin-2-yl]-hydrazonomethyl}-benzenesulfonicacid; and2-{[5-(4-{[(6-Hydroxycarbamoyl-7-isobutyl-8-oxo-2-oxa-9-aza-bicyclo[10.2.2]hexadeca-1(15),12(16),13-triene-10-carbonyl)-amino]-methyl}-benzylcarbamoyl)-pyridin-2-yl]-hydrazonomethyl}-benzenesulfonicacid; and wherein the cytotoxic radioisotope is ^(99m)Tc.
 11. Aradiopharmaceutical according to claim 1 wherein the cytotoxicradioisotope is selected from the group consisting of beta particleemitters, alpha particle emitters, and Auger electron emitters.
 12. Aradiopharmaceutical according to claim 1 wherein the cytotoxicradioisotope is selected from the group consisting of: ¹⁸⁶Re, ¹⁸⁸Re,¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁴⁹Pm, ⁹⁰Y, ²¹²Bi, ¹⁰³Pd, ¹⁰⁹Pd, ¹⁵⁹Gd, ¹⁴⁰La,¹⁹⁸Au, ¹⁹⁹Au, ¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁶⁵Dy, ¹⁶⁶Dy, ⁶⁷Cu, ¹⁰⁵Rh, ¹¹¹Ag, and ¹⁹²Ir.13. A radiopharmaceutical according to claim 1 wherein the cytotoxicradioisotope is selected from the group consisting of ¹⁸⁶Re, ¹⁸⁸Re,¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁴⁹Pm, ⁹⁰Y, ²¹²Bi, ¹⁰³Pd, and ¹⁰⁵Rh.
 14. Aradiopharmaceutical according to claim 1 wherein the cytotoxicradioisotope is selected from the group consisting of: ¹⁸⁶Re, ¹⁸⁸Re,¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁴⁹Pm, ⁹⁰Y, and ²¹²Bi.
 15. A radiopharmaceuticalcomposition comprising a radiopharmaceutical of claim 1, or apharmaceutical acceptable salt thereof, and a pharmaceuticallyacceptable carrier.
 16. A radiopharmaceutical kit comprising aradiopharmaceutical of claim 1, or a pharmaceutical acceptable salt formthereof and a pharmaceutically acceptable carrier.
 17. Aradiopharmaceutical kit of claim 16 further comprising a stabilizer. 18.A radiopharmaceutical kit according to claim 16, wherein theradioisotope is ¹⁸⁶Re or ¹⁸⁸Re and the kit further comprises one or moreancillary ligands and a reducing agent.
 19. A radiopharmaceutical kitaccording to claim 18, wherein the ancillary ligands are tricine and aphosphine.
 20. A method of treating a pathological disorder mediated bya matrix metalloproteinase in a patient which comprises administering toa patient in need thereof a therapeutically effective amount of aradiopharmaceutical according to claim 1 and a pharmaceuticallyacceptable carrier.
 21. A method of inhibiting proliferation of cancercells, comprising contacting the cancer cells with aproliferation-inhibitory amount of a radiopharmaceutical of claim
 1. 22.A method of claim 20, wherein the matrix metalloproteinase is selectedfrom the group consisting of: MMP-1, MMP-2, MMP-3, MMP-9, and MMP-14.23. A method of claim 20 wherein the matrix metalloproteinase isselected from the group consisting of: MMP-2, MMP-9, and MMP-14.
 24. Aprocess for the preparation of a radiopharmaceutical, said processcomprising generating a macrostructure from a plurality of molecularcomponents wherein the plurality of components comprises aradiopharmaceutical according to claim.